Method for analyzing microbial community structure based on reverse transcription of full-length small subunit ribosomal RNA

Abstract

The invention relates to a method for analyzing a microbial community structure based on reverse transcription of full-length small subunit ribosomal RNA. The method comprises the following steps: (1)rapidly extracting total RNA in a sample; (2) adding a first linker sequence at the 5'end of the RNA; (3) adding a second linker sequence at the 5'end of the universal reverse primer capable of covering eukaryotes and prokaryotes for reverse transcription of small subunit ribosomal RNA; (4) screening ribosome SSU cDNA in a reverse transcription product; (5) carrying out PCR amplification to obtain double-stranded full-length ribosome SSU cDNA, sequencing an amplification product, and extracting a full-length SSU rRNA sequence; and (6) determining accurate classification information accordingto the obtained full-length SSU rRNA sequence to obtain a fine microbial community structure. Compared with the prior art, the method has the advantages that a universal forward primer is not used, deviation generated in the PCR process is reduced, microorganisms omitted in PCR amplification of a conventional universal primer can be obtained, and the overall community structure and the like of bacteria, archaea and eukaryotic microorganisms are analyzed at the same time.

Description

technical field [0001] The invention belongs to the technical field of biological analysis, and relates to a method for analyzing microbial community structure based on reverse transcription of full-length small subunit ribosomal RNA. Background technique [0002] Isolation and culture is a traditional technique to study the species and composition of environmental microorganisms, but most of the microorganisms cannot be isolated in laboratory conditions, and the research of environmental microorganisms has encountered a bottleneck. With the development of sequencing technology, especially the popularization of next-generation sequencing technology, scientists can study the classification and composition of environmental microorganisms through culture-independent techniques. A commonly used technique is to amplify the SSU rRNA gene through "universal" primers, high-throughput sequencing, and through subsequent sequence processing and analysis, to uncover the community struct...

AI Technical Summary

Problems solved by technology

However, the "universal" primers used in this method suffer from bias and coverage issues, resulting in amplification that favors certain classes of microorganisms or misses some microorganisms

For example, the modified 515F / 806R in the Earth Microbiome Project is our most commonly used primer for analyzing environmental microbial diversity, but it still has a 9.6% mismatch rate in primer evaluation for metagenomic data (loe-Fadrosh, E.,Ivanova,N.,Woyke,T.et al.(2016).Metagenomics uncovers gaps in amplicon-based detection of microbial diversity.Nat Microbiol 1,15032.); In addition, with the development of high-throughput sequencing technology, The second-generation high-throughput sequencing platform is commonly used for these amplified products, and its read length does not exceed 600bp, and the classification of the obtained SSU rRNA gene sequences is not accurate enough; moreover, routine microbial community structure analysis is generally aimed at extracting from samples DNA is amplified by PCR, which cannot distinguish between real living microorganisms and dead microorganisms in the environment

[0003]In previous studies, Li Xiaoran et al. (Li,X.R.,Lv,Y.,Meng,H.,Gu,J.D.,and Quan,Z.X.(2014). Analysis of microbial diversity by pyrosequencing the small-subunitribosomal RNA without PCR amplification. Appl Microbiol Biotechnol 98(9), 3777-3789.) By analyzing the community structure of SROP (Small-subunit Ribosomal RNA withOut specific PCR amplification), avoiding The influence of "universal" primers can identify active microorganisms in the community and improve the accuracy of community structure analysis (related patent number: ZL201010132091.6), but the extremely high amount of starting microbial RNA, the sequence start site is not fixed and The length of the taxonomic sequence restricts its wide application; Yan Yongwei et al. rRNA in different types of environmental samples.PLoS One 12(10), e0186161.) Reverse transcribe SSU rRNA with random primers adding another adapter after adding an adapter at the 5' end of the RNA, and design primers based on the adapter sequences at both ends The method of PCR amplification further solves the problem that the transcriptome analysis community structure requires high starting RNA and the sequence start site is not fixed, but the length of the available classification sequence is still not more than 600bp

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