Microbial Ecology

, Volume 65, Issue 3, pp 709–719 | Cite as

Effects of OTU Clustering and PCR Artifacts on Microbial Diversity Estimates

  • Nastassia V. Patin
  • Victor Kunin
  • Ulrika Lidström
  • Matthew N. Ashby
Methods

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.

Supplementary material

248_2012_145_MOESM1_ESM.docx (101 kb)
ESM 1(DOCX 101 kb)

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Copyright information

© Springer Science+Business Media New York 2012

Authors and Affiliations

  • Nastassia V. Patin
    • 1
    • 3
  • Victor Kunin
    • 2
  • Ulrika Lidström
    • 2
  • Matthew N. Ashby
    • 1
    • 2
  1. 1.Romberg Tiburon Center for Environmental StudiesSan Francisco State UniversityTiburonUSA
  2. 2.Taxon Biosciences, Inc.TiburonUSA
  3. 3.Center for Marine Biotechnology and Biomedicine, Scripps Institution of OceanographyUniversity of California, San DiegoLa JollaUSA

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