Unité d'Ecologie, Systématique et Evolution, CNRS UMR 8079, Université Paris-Sud, Orsay, France.
ISME J. 2013 Feb;7(2):244-55. doi: 10.1038/ismej.2012.106. Epub 2012 Oct 4.
Deep-sequencing technologies are becoming nearly routine to describe microbial community composition in environmental samples. The 18S ribosomal DNA (rDNA) pyrosequencing has revealed a vast diversity of infrequent sequences, leading to the proposition of the existence of an extremely diverse microbial 'rare biosphere'. Although rare microbes no doubt exist, critical views suggest that many rare sequences may actually be artifacts. However, information about how diversity revealed by molecular methods relates to that revealed by classical morphology approaches is practically nonexistent. To address this issue, we used different approaches to assess the diversity of tintinnid ciliates, a species-rich group in which species can be easily distinguished morphologically. We studied two Mediterranean marine samples with different patterns of tintinnid diversity. We estimated tintinnid diversity in these samples employing morphological observations and both classical cloning and sequencing and pyrosequencing of two different markers, the 18S rDNA and the internal transcribed spacer (ITS) regions, applying a variety of computational approaches currently used to analyze pyrosequence reads. We found that both molecular approaches were efficient in detecting the tintinnid species observed by microscopy and revealed similar phylogenetic structures of the tintinnid community at the species level. However, depending on the method used to analyze the pyrosequencing results, we observed discrepancies with the morphology-based assessments up to several orders of magnitude. In several cases, the inferred number of operational taxonomic units (OTUs) largely exceeded the total number of tintinnid cells in the samples. Such inflation of the OTU numbers corresponded to 'rare biosphere' taxa, composed largely of artifacts. Our results suggest that a careful and rigorous analysis of pyrosequencing data sets, including data denoising and sequence clustering with well-adjusted parameters, is necessary to accurately describe microbial biodiversity using this molecular approach.
高通量测序技术已广泛应用于环境样本中微生物群落组成的描述。18S 核糖体 DNA（rDNA）焦磷酸测序揭示了大量罕见序列的存在，这导致了微生物“稀有生物圈”的存在假设。尽管稀有微生物无疑存在，但有批判性的观点认为，许多罕见序列实际上可能是人为假象。然而，关于分子方法所揭示的多样性与经典形态学方法所揭示的多样性之间的关系的信息实际上是不存在的。为了解决这个问题，我们使用了不同的方法来评估纤毛虫的多样性，纤毛虫是一类物种丰富的群体，其物种可以通过形态学很容易地区分。我们研究了两个具有不同纤毛虫多样性模式的地中海海洋样本。我们通过形态学观察以及使用两种不同的标记物（18S rDNA 和内部转录间隔区（ITS）区域）进行经典克隆和测序以及焦磷酸测序，采用目前用于分析焦序列读数的各种计算方法来估计这些样本中的纤毛虫多样性。我们发现，两种分子方法都能有效地检测到通过显微镜观察到的纤毛虫物种，并揭示了在物种水平上纤毛虫群落的相似系统发育结构。然而，根据用于分析焦磷酸测序结果的方法，我们观察到与基于形态学的评估结果存在差异，甚至达到几个数量级。在某些情况下，推断的操作分类单元（OTU）数量大大超过了样本中纤毛虫细胞的总数。这种 OTU 数量的膨胀对应于主要由人为假象组成的“稀有生物圈”分类单元。我们的结果表明，需要对焦磷酸测序数据集进行仔细和严格的分析，包括使用适当调整参数的数据去噪和序列聚类，以便使用这种分子方法准确地描述微生物生物多样性。
