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Appl Environ Microbiol. 2014 Jul;80(14):4363-73. doi: 10.1128/AEM.00057-14. Epub 2014 May 9.

Investigating microbial eukaryotic diversity from a global census: insights from a comparison of pyrotag and full-length sequences of 18S rRNA genes.

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Department of Biological Sciences, University of Southern California, Los Angeles, California, USA
Department of Biological Sciences, University of Southern California, Los Angeles, California, USA.
Bigelow Laboratory for Ocean Sciences, East Boothbay, Maine, USA.
The Josephine Bay Paul Center for Comparative Molecular Biology and Evolution, Marine Biological Laboratory, Woods Hole, Massachusetts, USA Department of Geological Sciences, Brown University, Providence, Rhode Island, USA.
Environmental Research Institute, School of Science, University of Waikato, Hamilton, New Zealand College of Earth, Ocean, and Environment, University of Delaware, Lewes, Delaware, USA.
College of Oceanic and Atmospheric Sciences, Oregon State University, Corvallis, Oregon, USA.
Woods Hole Oceanographic Institution, Woods Hole, Massachusetts, USA.


Next-generation DNA sequencing (NGS) approaches are rapidly surpassing Sanger sequencing for characterizing the diversity of natural microbial communities. Despite this rapid transition, few comparisons exist between Sanger sequences and the generally much shorter reads of NGS. Operational taxonomic units (OTUs) derived from full-length (Sanger sequencing) and pyrotag (454 sequencing of the V9 hypervariable region) sequences of 18S rRNA genes from 10 global samples were analyzed in order to compare the resulting protistan community structures and species richness. Pyrotag OTUs called at 98% sequence similarity yielded numbers of OTUs that were similar overall to those for full-length sequences when the latter were called at 97% similarity. Singleton OTUs strongly influenced estimates of species richness but not the higher-level taxonomic composition of the community. The pyrotag and full-length sequence data sets had slightly different taxonomic compositions of rhizarians, stramenopiles, cryptophytes, and haptophytes, but the two data sets had similarly high compositions of alveolates. Pyrotag-based OTUs were often derived from sequences that mapped to multiple full-length OTUs at 100% similarity. Thus, pyrotags sequenced from a single hypervariable region might not be appropriate for establishing protistan species-level OTUs. However, nonmetric multidimensional scaling plots constructed with the two data sets yielded similar clusters, indicating that beta diversity analysis results were similar for the Sanger and NGS sequences. Short pyrotag sequences can provide holistic assessments of protistan communities, although care must be taken in interpreting the results. The longer reads (>500 bp) that are now becoming available through NGS should provide powerful tools for assessing the diversity of microbial eukaryotic assemblages.

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