Method for constructing bacterium 16S rDNA overall-length high-throughput sequencing library

Abstract

The invention relates to a method for constructing a bacterium 16S rDNA overall-length high-throughput sequencing library, and relates to the field of microorganism high-throughput sequencing. The method comprises the steps of firstly, adding unique molecular identifiers (UMIs) which are composed of random base groups to the two ends of each 16S rDNA template molecule, conducting PCR amplificationto obtain multiple copied 16S rDNA overall-length amplicons with two ends containing specific UMIs, randomly breaking and connecting joints of the sequencing library, and conducting paired-end sequencing to obtain the UMI sequences and corresponding 16S rDNA fragment sequences; besides, through cyclizing of the 16S rDNA overall-length amplicons and sequencing, obtaining UMI pairing information oftwo ends of the same molecule; conducting reads assembly according to the same UMI and pairing UMI information to increase the splicing length accordingly, and finally conducting assembly to obtain 16S rDNA overall-length sequence information. The method is low in cost, high in accuracy and suitable for high-throughput sequencing platforms; meanwhile, the method can effectively eliminate the influence of PCR amplification preferences, amplification errors and sequencing errors through the UMIs, and thus the bacterium population abundance in a sample is more precisely quantified.

Description

technical field [0001] The invention belongs to the field of high-throughput sequencing of microbial genes, in particular to a method for constructing a full-length high-throughput sequencing library of bacterial 16SrDNA. Background technique [0002] 16S ribosomal ribonucleic acid (ribosomal RNA, rRNA) is a subunit of ribosomal RNA, 16S rDNA is the gene encoding this subunit, exists in all bacterial genomes, is about 1.5Kb in length, and its molecular size is moderate, mutation It is the most commonly used and useful symbol in the study of bacterial taxonomy. 16S rDNA includes 9 variable regions and 10 conserved regions. The sequence of the conserved region reflects the relationship between species, while the sequence of the variable region can reflect the difference between species. 16S rDNA is often used to study species information in specific environments, including species differences, species classification and species abundance, etc., and plays an important guiding ...

AI Technical Summary

Problems solved by technology

However, full-length sequencing has more advantages. At present, the full-length sequencing of 16S rDNA mainly uses third-generation sequencing, such as Pacbio platform sequencing, which has the advantage of longer read length and can obtain the sequence information of the full-length 16S rDNA. Small

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