Pyrosequencing for assessing diversity of eukaryotic microbes: analysis of data on marine planktonic ciliates and comparison with traditional methods.
Pyrosequencing for assessing diversity of eukaryotic microbes: analysis of data on marine planktonic ciliates and comparison with traditional methods.
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DOI：
10.1111/1462-2920.12380
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发表时间：
2014-09
影响因子：
5.1
通讯作者：
L. Santoferrara;Jean-David Grattepanche;L. Katz;G. McManus
中科院分区：
文献类型：
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作者：
L. Santoferrara;Jean-David Grattepanche;L. Katz;G. McManus
评估微生物多样性需要分析所有三个生命领域，包括真核微生物。我们研究了两个生态学上重要的分支的微生物真核生物，纤毛虫的亚类寡毛和Choreotrichia（类Spirotrichea）的多样性，通过比较焦磷酸测序桑格测序克隆库和显微镜。使用从一个大的温带河口（长岛湾，美国）的样本，我们得到了三个主要的见解。首先，丰富的估计变化高达一个数量级，无论是使用不同的标准焦磷酸测序处理或焦磷酸测序，克隆和显微镜，而分类鉴定几乎总是一致的。用于pyrosequences的纠错算法（“去噪”）减少了丰富度的差异，但也从数据中删除了已知的形态种。第二，虽然大多数的pyrosequenced的操作分类单位（OTU）分布在已知的订单和家庭，我们发现的证据，以前的特点或未知的分支，即使在这些纤毛虫谱系有丰富的历史形态描述。第三，焦磷酸测序允许检测在不同样品中占优势或极其罕见的OTU。我们的研究结果证实了焦磷酸测序定量微生物多样性的潜力，但也强调了使用这种方法来解决生态问题的焦磷酸测序处理仔细评估的重要性。
Assessing microbial diversity requires analysis of all three domains of life, including eukaryotic microbes. We examined the diversity of two ecologically important clades of microbial eukaryotes, ciliates in the subclasses Oligotrichia and Choreotrichia (class Spirotrichea), by comparing pyrosequencing to Sanger-sequenced clone libraries and microscopy. Using samples from a large temperate estuary (Long Island Sound, USA), we gained three major insights. First, richness estimates varied by up to one order of magnitude either using different criteria for pyrosequence processing or among pyrosequencing, cloning and microscopy, while taxon identification was almost always coherent. Error-correcting algorithms for pyrosequences ('denoising') reduced discrepancies in richness but also removed known morphospecies from the data. Second, although most of the pyrosequenced operational taxonomic units (OTUs) were distributed within known orders and families, we found evidence of a previously uncharacterized or unknown clade even in these ciliate lineages that have a rich history of morphological description. Third, pyrosequencing allowed the detection of OTUs that were either dominant or extremely rare in different samples. Our findings confirm the potential of pyrosequencing for quantifying microbial diversity, but also highlight the importance of careful evaluation of pyrosequence processing for using this method to address ecological questions.