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Primer set for building 16S rRNA gene amplicon sequencing library and building method

A sequencing library and gene amplification technology, applied in biochemical equipment and methods, DNA/RNA fragments, chemical libraries, etc., can solve the problem of low sequence base complexity, improve sequencing quality, optimize library construction methods, The effect of reducing the cost of experiments

Active Publication Date: 2018-03-23
湖南赛哲智造科技有限公司
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AI Technical Summary

Problems solved by technology

[0006] In order to overcome the above-mentioned deficiencies of the prior art, the present invention provides a quadruple index primer set for constructing a 16S rRNA gene amplicon sequencing library, which fully utilizes the characteristics of the next-generation sequencing technology and the amplicon library composed of microbial communities Features, breaking through the inherent limitations of sequencing platforms to solve problems such as low sequence base complexity

Method used

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  • Primer set for building 16S rRNA gene amplicon sequencing library and building method
  • Primer set for building 16S rRNA gene amplicon sequencing library and building method
  • Primer set for building 16S rRNA gene amplicon sequencing library and building method

Examples

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Effect test

Embodiment 1 4

[0053] Example 1 Design of four-fold label primer

[0054] 1. The first step PCR double label primer pair design

[0055] 1. Target primer design

[0056] Taking the V4-V5 region of the 16S rRNA gene as the target, a target primer pair was designed, including the upstream target primer Primer-F and the downstream target primer Primer-R, for the amplification of the target sequence. The specific design requirements are: (1) It is located in the gene conservative region and is subject to less variation among different bacterial species; (2) The base is balanced, the primers are specific, and the target band can be accurately amplified.

[0057] Correction primers based on design requirements and representative bacterial group 16S genome database, such as common fecal gram-positive bacteria Clostridioides difficile 630, complete genome (ACCESSION: CP010905) and gram-negative bacteria Escherichia coli 16S ribosomal RNA, complete sequence(ACCESSION: J01859). After extensive verification ...

Embodiment 2

[0132] A method for constructing a 16S rRNA gene amplicon sequencing library, which mainly includes the following steps: sample genome extraction and quality control, adding any of the four-label primer sets described in Example 1, the first step of PCR reaction and product detection, In the second step, PCR reaction and product detection, library quantification and magnetic bead purification, add 5% PiX library to the 16S rRNA gene amplicon sequencing library for computer sequencing. Each sample needs to use the first PCR double-label primer pair for target amplification, and the second PCR double-label primer pair for complementary sequencing adapters. After a total of two rounds of PCR are completed, the test is qualified, and the complete 16S rRNA gene amplicon sequencing library. Specific steps are as follows:

[0133] 1. Fecal genome extraction

[0134] (1) Genome extraction

[0135] The genomic DNA of human feces was extracted by the kit (OMEGA cat.D4015). In order to ensu...

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Abstract

The invention discloses a quadruple-tag primer set for building a 16S rRNA gene amplicon sequencing library. The quadruple-tag primer set is composed of a first-step PCR double-tag primer pair and a second-step PCR double-tag primer pair, the sequence of an upstream primer of the first-step PCR double-tag primer pair is one of SEQ ID NO:1-8 while that of a downstream primer of the same is one of SEQ ID NO:9-20, and the sequence of an upstream primer of the second-step PCR double-tag primer pair is one of SEQ ID NO:21-44 while that of a downstream primer of the same is one of SEQ ID NO:45-68. Abalanced base sequence is added into the quadruple-tag primer, so that diversity of the library is improved, and sequencing quality is improved; during online sequencing, only a small amount of PhiXlibrary needs to be mixed, and staggered sequencing needs to be performed, so that waste of sequencing flux is reduced, and sequencing quality is ensured.

Description

Technical field [0001] The present invention relates to the technical field of microbial gene high-throughput sequencing, in particular to a primer set and a construction method for constructing a 16SrRNA gene amplicon sequencing library. Background technique [0002] With the continuous improvement of sequencing technology, microbial gene high-throughput sequencing technology has become increasingly mature. Amplicon sequencing is a targeted sequencing method for polymorphic gene regions (such as 16S, 18S, ITS genes) that hold biological genetic information. During the process of library construction, the target region needs to be amplified first. Then the PCR products were constructed and sequenced. 16SrRNA sequencing can evaluate the composition of multi-sample microbial populations at a low cost, and is almost unaffected by host genome contamination, but it also has the following shortcomings: (1) The sequencing information area is less, and it is used in the identification a...

Claims

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Application Information

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IPC IPC(8): C40B50/06C12N15/11C12Q1/6869
CPCC12N15/1093C12N15/11C12Q1/6869C40B50/06C12Q2535/122C12Q2531/113C12Q2537/143
Inventor 伍泳彰陈杰
Owner 湖南赛哲智造科技有限公司
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