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Effects of OTU Clustering and PCR Artifacts on Microbial Diversity Estimates

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Abstract

Next-generation sequencing has increased the coverage of microbial diversity surveys by orders of magnitude, but differentiating artifacts from rare environmental sequences remains a challenge. Clustering 16S rRNA sequences into operational taxonomic units (OTUs) organizes sequence data into groups of 97 % identity, helping to reduce data volumes and avoid analyzing sequencing artifacts by grouping them with real sequences. Here, we analyze sequence abundance distributions across environmental samples and show that 16S rRNA sequences of >99 % identity can represent functionally distinct microorganisms, rendering OTU clustering problematic when the goal is an accurate analysis of organism distribution. Strict postsequencing quality control (QC) filters eliminated the most prevalent artifacts without clustering. Further experiments proved that DNA polymerase errors in polymerase chain reaction (PCR) generate a significant number of substitution errors, most of which pass QC filters. Based on our findings, we recommend minimizing the number of PCR cycles in DNA library preparation and applying strict postsequencing QC filters to reduce the most prevalent artifacts while maintaining a high level of accuracy in diversity estimates. We further recommend correlating rare and abundant sequences across environmental samples, rather than clustering into OTUs, to identify remaining sequence artifacts without losing the resolution afforded by high-throughput sequencing.

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Acknowledgments

We thank Stewart Scherer for his bioinformatics assistance and helpful comments.

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Author notes

  1. Nastassia V. Patin

    Present address: Center for Marine Biotechnology and Biomedicine, Scripps Institution of Oceanography, University of California, San Diego, La Jolla, CA, 92093-0204, USA

Authors and Affiliations

  1. Romberg Tiburon Center for Environmental Studies, San Francisco State University, 3152 Paradise Drive, Tiburon, CA, 94920, USA

    Nastassia V. Patin & Matthew N. Ashby

  2. Taxon Biosciences, Inc., 3150 Paradise Drive, Tiburon, CA, 94920, USA

    Victor Kunin, Ulrika Lidström & Matthew N. Ashby

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  1. Nastassia V. Patin
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  2. Victor Kunin
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Correspondence to Nastassia V. Patin.

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Patin, N.V., Kunin, V., Lidström, U. et al. Effects of OTU Clustering and PCR Artifacts on Microbial Diversity Estimates. Microb Ecol 65, 709–719 (2013). https://doi.org/10.1007/s00248-012-0145-4

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  • DOI: https://doi.org/10.1007/s00248-012-0145-4

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