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Applied and Environmental Microbiology
Vol. xx, No. xx, 20
14; Page: xx–xx

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

Alle A.Y. Lie, Zhenfeng Liu, Sarah K. Hu, Adriane C. Jones, Diane Y. Kim, Peter D. Countway, Linda A. Amaral-Zettler, S. Craig Cary, Evelyn B. Sherr, Barry F. Sherr, Rebecca J. Gast and David A. Caron

Department of Biological Sciences, University of Southern California, 3616 Trousdale Parkway, Los Angeles, California 90089-0371, USA.

Abstract

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 gene from ten global samples were analyzed in order to compare the resulting protistan community structure and species richness. Pyrotag OTUs called at 98% sequence similarity yielded similar overall numbers of OTUs as full-length sequences when the latter were called at 97% similarity. Singleton OTUs strongly influenced estimates of species richness but not higher-level taxonomic composition of the community. The pyrotag and full-length sequence dataset had slightly different taxonomic composition of rhizarians, stramenopiles, cryptophytes, and haptophytes, but both datasets had similarly high composition of alveolates. Pyrotag OTUs with the most pyrotag sequences were comprised of pyrotags that mapped at 100% similarity to multiple full-length OTUs. 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 datasets yielded similar clusters, indicating that beta diversity analysis results were similar for both Sanger and NGS sequences. Short pyrotag sequences can provide holistic assessments of protistan communities, although care must be taken when interpreting the results. Longer reads (> 500 bp) now becoming available through NGS should provide powerful tools for assessing the diversity of microbial eukaryotic assemblages.

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