Analysis of run-to-run variation of bar-coded pyrosequencing for evaluating bacterial community shifts and individual taxa dynamics

PLoS One. 2014 Jun 9;9(6):e99414. doi: 10.1371/journal.pone.0099414. eCollection 2014.


Bar-coded pyrosequencing has been increasingly used due to its fine taxonomic resolution and high throughput. Yet, concerns arise regarding the reproducibility of bar-coded pyrosequencing. We evaluated the run-to-run variation of bar-coded pyrosequencing in detecting bacterial community shifts and taxa dynamics. Our results demonstrate that pyrosequencing is reproducible in evaluating community shifts within a run, but not between runs. Also, the reproducibility of pyrosequencing in detecting individual taxa increased as a function of taxa abundance. Based on our findings: (1) for studies with modest sequencing depth, it is doubtful that data from different pyrosequencing runs can be considered comparable; (2) if multiple pyrosequencing runs are needed to increase the sequencing depth, additional sequencing efforts should be applied to all samples, rather than to selected samples; (3) if pyrosequencing is used for estimating bacterial population dynamics, only the abundant taxa should be considered; (4) for less-abundant taxa, the sequencing depth should be increased to ensure an accurate evaluation of taxon variation trends across samples.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Analysis of Variance
  • Bacteria / classification*
  • Bacteria / genetics*
  • Biodiversity
  • DNA Barcoding, Taxonomic*
  • High-Throughput Nucleotide Sequencing* / methods
  • High-Throughput Nucleotide Sequencing* / standards
  • Metagenome*
  • Reproducibility of Results
  • Soil Microbiology

Grant support

This work was supported by the National Science Foundation and the Environmental Protection Agency under Cooperative Agreement DBI-0830117. Any opinions, findings, and conclusions are those of the authors and do not necessarily reflect the views of the National Science Foundation or the Environmental Protection Agency. This work has not been subjected to EPA review and no official endorsement should be inferred. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.