Ge Yuan, Schimel Joshua P, Holden Patricia A
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.
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.
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.
由于其精细的分类分辨率和高通量，条形码焦磷酸测序技术的应用越来越广泛。然而，条形码焦磷酸测序的可重复性引发了人们的关注。我们评估了条形码焦磷酸测序在检测细菌群落变化和分类群动态方面的批次间差异。我们的结果表明，焦磷酸测序在评估单次运行内的群落变化时具有可重复性，但在不同运行之间则不然。此外，焦磷酸测序在检测单个分类群时的可重复性随着分类群丰度的增加而提高。基于我们的研究结果：（1）对于测序深度适中的研究，不同焦磷酸测序运行的数据是否具有可比性值得怀疑；（2）如果需要多次焦磷酸测序运行以增加测序深度，应将额外的测序工作应用于所有样本，而不是选定的样本；（3）如果使用焦磷酸测序来估计细菌种群动态，仅应考虑丰富的分类群；（4）对于丰度较低的分类群，应增加测序深度以确保准确评估样本间分类群的变化趋势。
