title: Analysis of run-to-run variation of bar-coded pyrosequencing for evaluating bacterial community shifts and individual taxa dynamics.
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.
authors.affiliation: 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.
authors.forename: Yuan
authors.initials: Y
authors.lastname: Ge
authors.affiliation: 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.
authors.forename: Joshua P
authors.initials: JP
authors.lastname: Schimel
authors.affiliation: 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.
authors.forename: Patricia A
authors.initials: PA
authors.lastname: Holden
mesh_terms: D000704:Analysis of Variance; D001419:Bacteria / Q000145:classification* / Q000235:genetics*; D044822:Biodiversity; D058893:DNA Barcoding, Taxonomic; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods / Q000592:standards; D054892:Metagenome; D015203:Reproducibility of Results; D012988:Soil Microbiology