How Much Do rRNA Gene Surveys Underestimate Extant Bacterial Diversity?
How Much Do rRNA Gene Surveys Underestimate Extant Bacterial Diversity?
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DOI：
10.1128/aem.00014-18
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发表时间：
2018-03-01
影响因子：
4.4
通讯作者：
Konstantinidis, Konstantinos T.
中科院分区：
文献类型：
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作者：
Rodriguez-R, Luis M.;Castro, Juan C.;Konstantinidis, Konstantinos T.
研究和编目原核生物多样性最常见的做法是在97%的16S rRNA基因序列同源性水平上将序列分组为可操作的分类单元(OTU)，通常使用部分基因序列，如聚合酶链式反应产生的扩增片段。由于rRNA基因的高度序列保守性，属于密切相关但不同物种的生物可能被归类到同一OTU下。然而，目前尚不清楚这种做法在多大程度上低估了多样性。为了解决这个问题，我们比较了定义在97%或98.5%16S rRNA基因同源性水平上的基因组的OTUS和定义在95%全基因组平均核苷酸同源性(ANI)的相同基因组的OTUS，ANI是一个更准确的物种替代指标。我们的结果表明，与95%的ANI(90.5%对89.9%)相比，98.5%的16S rRNA基因同源性截断值所产生的OTUS的准确率高于97%，但与98.29-98.78%范围内的任何其他阈值没有区别。然而，即使有更严格的阈值，与基于ANI的方法相比，基于16S rRNA基因的方法通常通常低估OTU的数量，平均约为12%(当使用97%的同一性阈值时，类似于14%的低估)。更重要的是，对于某些分类群，如假单胞菌属、伯克霍尔德氏菌属、埃希里希菌属、弯曲杆菌属和柠檬酸杆菌属，低估程度可能达到50%或更多。这些结果提供了16S rRNA基因定义的OTUS低估现有原核生物多样性的程度的定量视图，并表明基因组解析通常是必要的。重要的物种多样性是群落生态学和保护生物学最基本的信息之一。因此，采用准确的物种或多样性单位的替代物是大量微生物生态学和多样性研究的基石。目前使用的最常见的指标依赖于16S rRNA基因序列在某个核苷酸同源性阈值下的聚类，通常为97%或98.5%。在这里，我们探索这一策略如何很好地反映更准确的基于全基因组的代理，并确定16S rRNA序列的高度保守掩盖了物种水平的实质性多样性的频率。
The most common practice in studying and cataloguing prokaryotic diversity involves the grouping of sequences into operational taxonomic units (OTUs) at the 97% 16S rRNA gene sequence identity level, often using partial gene sequences, such as PCR-generated amplicons. Due to the high sequence conservation of rRNA genes, organisms belonging to closely related yet distinct species may be grouped under the same OTU. However, it remains unclear how much diversity has been underestimated by this practice. To address this question, we compared the OTUs of genomes defined at the 97% or 98.5% 16S rRNA gene identity level against OTUs of the same genomes defined at the 95% whole-genome average nucleotide identity (ANI), which is a much more accurate proxy for species. Our results show that OTUs resulting from a 98.5% 16S rRNA gene identity cutoff are more accurate than 97% compared to 95% ANI (90.5% versus 89.9% accuracy) but indistinguishable from any other threshold in the 98.29 to 98.78% range. Even with the more stringent thresholds, however, the 16S rRNA gene-based approach commonly underestimates the number of OTUs by similar to 12%, on average, compared to the ANI-based approach (similar to 14% underestimation when using the 97% identity threshold). More importantly, the degree of underestimation can become 50% or more for certain taxa, such as the genera Pseudomonas, Burkholderia, Escherichia, Campylobacter, and Citrobacter. These results provide a quantitative view of the degree of underestimation of extant prokaryotic diversity by 16S rRNA gene-defined OTUs and suggest that genomic resolution is often necessary.IMPORTANCE Species diversity is one of the most fundamental pieces of information for community ecology and conservational biology. Therefore, employing accurate proxies for what a species or the unit of diversity is are cornerstones for a large set of microbial ecology and diversity studies. The most common proxies currently used rely on the clustering of 16S rRNA gene sequences at some threshold of nucleotide identity, typically 97% or 98.5%. Here, we explore how well this strategy reflects the more accurate whole-genome-based proxies and determine the frequency with which the high conservation of 16S rRNA sequences masks substantial species-level diversity.