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Evaluation of the reproducibility of amplicon sequencing with Illumina MiSeq platform

Author

Listed:
  • Chongqing Wen
  • Liyou Wu
  • Yujia Qin
  • Joy D Van Nostrand
  • Daliang Ning
  • Bo Sun
  • Kai Xue
  • Feifei Liu
  • Ye Deng
  • Yuting Liang
  • Jizhong Zhou

Abstract

Illumina’s MiSeq has become the dominant platform for gene amplicon sequencing in microbial ecology studies; however, various technical concerns, such as reproducibility, still exist. To assess reproducibility, 16S rRNA gene amplicons from 18 soil samples of a reciprocal transplantation experiment were sequenced on an Illumina MiSeq. The V4 region of 16S rRNA gene from each sample was sequenced in triplicate with each replicate having a unique barcode. The average OTU overlap, without considering sequence abundance, at a rarefaction level of 10,323 sequences was 33.4±2.1% and 20.2±1.7% between two and among three technical replicates, respectively. When OTU sequence abundance was considered, the average sequence abundance weighted OTU overlap was 85.6±1.6% and 81.2±2.1% for two and three replicates, respectively. Removing singletons significantly increased the overlap for both (~1–3%, p

Suggested Citation

  • Chongqing Wen & Liyou Wu & Yujia Qin & Joy D Van Nostrand & Daliang Ning & Bo Sun & Kai Xue & Feifei Liu & Ye Deng & Yuting Liang & Jizhong Zhou, 2017. "Evaluation of the reproducibility of amplicon sequencing with Illumina MiSeq platform," PLOS ONE, Public Library of Science, vol. 12(4), pages 1-20, April.
    • Handle: RePEc:plo:pone00:0176716
    DOI: 10.1371/journal.pone.0176716
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