Accuracy of protist diversity assessments: morphology compared with cloning and direct pyrosequencing of 18S rRNA genes and ITS regions using ... View Full Text


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Article Info

DATE

2012-10-04

AUTHORS

Charles Bachy, John R Dolan, Purificación López-García, Philippe Deschamps, David Moreira

ABSTRACT

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. More... »

PAGES

244-255

References to SciGraph publications

  • 2009-07-23. Microbial community structure in the North Pacific ocean in THE ISME JOURNAL: MULTIDISCIPLINARY JOURNAL OF MICROBIAL ECOLOGY
  • 2011-01-28. Removing Noise From Pyrosequenced Amplicons in BMC BIOINFORMATICS
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  • 2009-09. The 'rare biosphere': a reality check in NATURE METHODS
  • 2001-02. Unexpected diversity of small eukaryotes in deep-sea Antarctic plankton in NATURE
  • 2007-07-20. Accuracy and quality of massively parallel DNA pyrosequencing in GENOME BIOLOGY
  • 2001-02. Oceanic 18S rDNA sequences from picoplankton reveal unsuspected eukaryotic diversity in NATURE
  • 2004-06-28. RETRACTED ARTICLE: TREEFINDER: a powerful graphical analysis environment for molecular phylogenetics in BMC ECOLOGY AND EVOLUTION
  • 2009-08-09. Accurate determination of microbial diversity from 454 pyrosequencing data in NATURE METHODS
  • 2010-01-21. Experimental factors affecting PCR-based estimates of microbial species richness and evenness in THE ISME JOURNAL: MULTIDISCIPLINARY JOURNAL OF MICROBIAL ECOLOGY
  • 2011-03-10. Protistan microbial observatory in the Cariaco Basin, Caribbean. I. Pyrosequencing vs Sanger insights into species richness in THE ISME JOURNAL: MULTIDISCIPLINARY JOURNAL OF MICROBIAL ECOLOGY
  • 2006-02. Probing Diversity in the Plankton: Using Patterns in Tintinnids (Planktonic Marine Ciliates) to Identify Mechanisms in HYDROBIOLOGIA
  • Identifiers

    URI

    http://scigraph.springernature.com/pub.10.1038/ismej.2012.106

    DOI

    http://dx.doi.org/10.1038/ismej.2012.106

    DIMENSIONS

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    PUBMED

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    33 schema:description 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.
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    40 ITS region
    41 OTU numbers
    42 accuracy
    43 analysis
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    47 biodiversity
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    49 case study
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    51 cells
    52 ciliates
    53 classical cloning
    54 cloning
    55 community
    56 community composition
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    78 issues
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    91 morphology
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    94 number
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    96 operational taxonomic units
    97 order
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    99 parameters
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    101 phylogenetic structure
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    103 pyrosequencing
    104 rDNA
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    109 reads
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