Document Detail

Accuracy of protist diversity assessments: morphology compared with cloning and direct pyrosequencing of 18S rRNA genes and ITS regions using the conspicuous tintinnid ciliates as a case study.
MedLine Citation:
PMID:  23038176     Owner:  NLM     Status:  MEDLINE    
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.
Charles Bachy; John R Dolan; Purificación López-García; Philippe Deschamps; David Moreira
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Publication Detail:
Type:  Comparative Study; Journal Article; Research Support, Non-U.S. Gov't     Date:  2012-10-04
Journal Detail:
Title:  The ISME journal     Volume:  7     ISSN:  1751-7370     ISO Abbreviation:  ISME J     Publication Date:  2013 Feb 
Date Detail:
Created Date:  2013-01-22     Completed Date:  2013-11-14     Revised Date:  2014-02-04    
Medline Journal Info:
Nlm Unique ID:  101301086     Medline TA:  ISME J     Country:  England    
Other Details:
Languages:  eng     Pagination:  244-55     Citation Subset:  IM    
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MeSH Terms
Ciliophora / classification,  genetics*
Cloning, Molecular
Cluster Analysis
Computational Biology
DNA, Ribosomal Spacer / genetics*
Gene Library
Mediterranean Sea
RNA, Ribosomal, 18S / genetics*
Sequence Analysis, DNA
Water Microbiology
Reg. No./Substance:
0/DNA, Ribosomal Spacer; 0/RNA, Ribosomal, 18S

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine

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