Microbial Diversity Biased Estimation Caused by Intragenomic Heterogeneity and Interspecific Conservation of 16S rRNA Genes
Microbial Diversity Biased Estimation Caused by Intragenomic Heterogeneity and Interspecific Conservation of 16S rRNA Genes
复制标题
DOI：
10.1128/aem.02108-22
复制
发表时间：
2023-04-27
影响因子：
4.4
通讯作者：
Zhou，Ning-Yi
中科院分区：
文献类型：
--
作者：
Pan，Piaopiao;Gu，Yichao;Zhou，Ning-Yi
16S rRNA 基因已被广泛用作分子标记，用于探索各种环境中的进化关系和分析微生物组成。尽管它方便且普遍，但局限性是不可避免的。可变的拷贝数、基因组内异质性和低分类分辨率导致了微生物多样性估计的偏差。对24,248个完整原核生物基因组的分析表明，细菌中的16S rRNA基因拷贝数为1至37，古细菌中的16S rRNA基因拷贝数为1至5，并且在60%的原核生物基因组中观察到基因组内异质性，其中大多数低于1%。当使用全长或部分16S rRNA基因时，计算了由基因组内变异引起的微生物多样性的高估和种间保守引起的低估。结果表明，在 100% 阈值下，使用全长基因时，微生物多样性可能被高估高达 156.5%。基于 V4 至 V5 区域的分析引入了最低的高估率 (4.4%)，但在 97% 阈值下，其物种分辨率略低于其他可变区域。对于不同的可变区域，提出了适当的阈值而不是标准值 97%，以尽量减少将单个基因组分裂成多个簇并将不同物种集中到同一簇中的风险。本研究不仅更新了现有原核生物基因组的16S rRNA基因拷贝数和基因组内变异信息，而且阐明了用定量数据估计原核生物多样性的偏差，为微生物群落调查中扩增区域和聚类阈值的选择提供参考。重要性微生物多样性通常采用基于标记基因的方法进行分析，其中16S rRNA基因测序是应用最广泛的方法。然而，由于 16S rRNA 基因的基因组内变异和低分类分辨率，获得微生物多样性的准确估计仍然是一个挑战。对不断增加的原核基因组中使用 16S rRNA 基因估计原核生物多样性的偏差进行全面检查，凸显了根据具体研究目标选择测序区域和聚类阈值的重要性。
The 16S rRNA gene has been extensively used as a molecular marker to explore evolutionary relationships and profile microbial composition throughout various environments. Despite its convenience and prevalence, limitations are inevitable. Variable copy numbers, intragenomic heterogeneity, and low taxonomic resolution have caused biases in estimating microbial diversity. Here, analysis of 24,248 complete prokaryotic genomes indicated that the 16S rRNA gene copy number ranged from 1 to 37 in bacteria and 1 to 5 in archaea, and intragenomic heterogeneity was observed in 60% of prokaryotic genomes, most of which were below 1%. The overestimation of microbial diversity caused by intragenomic variation and the underestimation introduced by interspecific conservation were calculated when using full-length or partial 16S rRNA genes. Results showed that, at the 100% threshold, microbial diversity could be overestimated by as much as 156.5% when using the full-length gene. The V4 to V5 region-based analyses introduced the lowest overestimation rate (4.4%) but exhibited slightly lower species resolution than other variable regions under the 97% threshold. For different variable regions, appropriate thresholds rather than the canonical value 97% were proposed for minimizing the risk of splitting a single genome into multiple clusters and lumping together different species into the same cluster. This study has not only updated the 16S rRNA gene copy number and intragenomic variation information for the currently available prokaryotic genomes, but also elucidated the biases in estimating prokaryotic diversity with quantitative data, providing references for choosing amplified regions and clustering thresholds in microbial community surveys.IMPORTANCEMicrobial diversity is typically analyzed using marker gene-based methods, of which 16S rRNA gene sequencing is the most widely used approach. However, obtaining an accurate estimation of microbial diversity remains a challenge, due to the intragenomic variation and low taxonomic resolution of 16S rRNA genes. Comprehensive examination of the bias in estimating such prokaryotic diversity using 16S rRNA genes within ever-increasing prokaryotic genomes highlights the importance of the choice of sequencing regions and clustering thresholds based on the specific research objectives.