分析方法对细菌群落16S rRNA基因扩增测序分析结果的影响

Effects of Analysis Methods on the Analyzed Results of 16S rRNA Gene Amplicon Sequencing in Bacterial Communities

细菌16S rRNA基因扩增测序是当前环境微生物组学研究中应用最为广泛的方法之一。然而,测序序列最小分类单元的划分有多种方式,其对微生物多样性下游分析结果的影响还有待进一步探究。本研究通过提取5组环境样本(森林、农田、湿地土壤、湖泊沉积物和水体)的DNA进行16S rRNA基因扩增测序,对测序结果同时采用5种最小分类单元的划分方式(基于97%、98%、99%和100%序列相似性聚类的OTU以及基于DADA2算法得到的ASV)进行划分,比较分析最小分类单元划分方法对微生物群落多样性、组成、以及其与环境因子关联性分析造成的影响。结果表明,提高分类分辨率,能够获得更高的群落α多样性(Chao1和Shannon)和β多样性(P < 0.05),而相对于按序列相似性聚类的OTU,ASV方法会在一定程度上降低Chao1和PD指数。对于群落组成,分类单元的划分方式主要影响微生物组一些低丰度属(< 0.2%)的占比,而对较高的分类学水平(门水平)组成的影响较小。此外,冗余分析的结果表明,提高分类分辨率水平,能够使得环境因子对微生物群落能够获得更高的解释度,同时也会影响各环境因子对群落组成的解释度排序。总之,本研究明晰了最小分类单元的不同划分方式会对微生物组多样性、组成以及与环境因子的关联性造成的影响,为后续环境微生物组学研究提供了理论指导。

Bacterial 16S rRNA gene amplicon sequencing is one of the most widely used method in environmental microbiology.However,there are many ways to determine the minimum taxonomic unit of the sequencing reads,and its effect on the downstream analysis results of microbial diversity needs to be further explored.This study extracted DNA from 5 groups of environmental samples(forest soil,farmland soil,wetland soil,lake sediment,and lake water)for 16S rRNA gene amplicon sequencing,and based on different taxonomic resolutions,5 types of minimum taxonomic unit division method(Sequence clustering OTUs at 97%,98%,99%,100%similarity,and the DADA 2 algorithm to calculate ASVs)were used to compare and analyze the influences of minimum taxonomic unit division methods on microbial community diversity,composition and its correlation with environmental factors.The results showed that higher communityαdiversity(Chao1 and Shannon)andβdiversity(P<0.05)were obtained by increasing the taxonomic resolution.Compared with the OTU,which was clustering by sequence similarity,the ASV method reduced Chao1 and PD indices to some extent.For community composition,the division method of the taxonomic unit mainly affected the proportion of some low-abundance genera(<0.2%),but had a smaller effect on the composition of higher taxonomy level(phylum level).In addition,the results of redundant analysis demonstrated that increasing the level of taxonomic resolution would allow the environmental factors to have a higher interpretation of the microbial community,and a concurrentlyit also affected the order of interpretation degree of community composition by environmental factors. In sum,this study clarified the effectof different division methods of the minimum taxonomy unit on the diversity,composition and association with environmental factors,whichprovides theoretical guidance for the subsequent study of environmental microbiomics.