Amplicon primer and establishment method for establishing variable region sequencing library of microorganism bacterium 16s rDNA

Abstract

The invention relates to an amplicon primer and an establishment method for establishing a variable region sequencing library of microorganism bacterium 16s rDNA. The amplicon primer consists of a forward primer and a reverse primer, wherein the forward primer comprises a first connector sequence, a first sequencing primer sequence, a first random base sequence and a forward specific target sequence primer; the reverse primer comprises a second connector sequence, a tag sequence, a second sequencing primer sequence, a second random base sequence and a reverse specific target sequence primer. In addition, by adopting the primer, one-step method amplicon can be adopted to establish the variable region sequencing library of microorganism bacterium 16s rDNA. By adopting the amplicon primer and the establishment method, the complexity of the library can be increased, the sequencing quality can be ensured, and sequencing flux waste caused by addition of a PhiX library can be avoided; in addition, the amplicon primer is high in amplification efficiency, dimer is unlikely to be generated, and bacterium populations can be accurately and rapidly identified with low cost.

Description

technical field [0001] The invention relates to the field of microbial gene sequencing, in particular to an amplicon primer for constructing a second-generation sequencing library of a microbial bacterial 16s rDNA variable region and a method for constructing a bacterial 16s rDNA variable region second-generation sequencing library. Background technique [0002] The 16S rRNA gene is a gene conserved in the evolution of prokaryotic bacteria, but there are some hypervariable segments on the 16S rRNA gene. Sequencing of hypervariable segments to distinguish bacterial species. At present, the metagenomic sequencing of microbial 16S rDNA genes generally uses the next-generation sequencing method, especially the Illumina MiSeq and HiSeq sequencing platforms with high accuracy and throughput. [0003] The current method for constructing a library for a small number of target genes basically uses the amplicon library construction method. That is, at the same time as the specific g...

AI Technical Summary

Problems solved by technology

Because Illumina MiSeq, HiSeq 2000 / 2500, and HiSeq XFive / Ten sequence low-complexity libraries, such as PCR amplicon libraries or simple gene libraries, if they sequence each cycle, especially the first 5 cycles, the measured The ratio of A / C / G / T of the four bases is not close to 25% each, which will cause the sequencing system to fail to correct the data, which will eventually lead to poor sequencing quality or even sequencing failure

For these libraries, the solution is to mix a PhiX library with a base group ratio close to 25% in the original sequencing library to increase the complexity of the sequencing library

But this will cause a waste of sequencing data path

[0007] When using the Illumina sequencing platform to sequence the 16S library, use the 16S rDNA library constructed by the above two methods. Since the 16S rDNA library is constructed from amplicons with only one or a few pairs of specific primers, the sequencing library is complex. If the degree is not enough, you need to add the PhiX library, otherwise the sequencing quality will be seriously affected, or even the sequencing will fail

However, adding the PhiX library will occupy 75% of the sequencing throughput data, which greatly wastes the sequencing throughput

And increase the steps and cost of 16S rDNA sequencing

Images