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

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

Author information

1
Earth Research Institute, University of California Santa Barbara, Santa Barbara, California, United States of America; Bren School of Environmental Science and Management, University of California Santa Barbara, Santa Barbara, California, United States of America; University of California Center for Environmental Implications of Nanotechnology, University of California Santa Barbara, Santa Barbara, California, United States of America.
2
University of California Center for Environmental Implications of Nanotechnology, University of California Santa Barbara, Santa Barbara, California, United States of America; Department of Ecology, Evolution and Marine Biology, University of California Santa Barbara, Santa Barbara, California, United States of America.

Abstract

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.

PMID:
24911191
PMCID:
PMC4049813
DOI:
10.1371/journal.pone.0099414
[Indexed for MEDLINE]
Free PMC Article

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