Assessing the fecal microbiota: an optimized ion torrent 16S rRNA gene-based analysis protocol

PLoS One. 2013 Jul 15;8(7):e68739. doi: 10.1371/journal.pone.0068739. Print 2013.

Abstract

Assessing the distribution of 16S rRNA gene sequences within a biological sample represents the current state-of-the-art for determination of human gut microbiota composition. Advances in dissecting the microbial biodiversity of this ecosystem have very much been dependent on the development of novel high-throughput DNA sequencing technologies, like the Ion Torrent. However, the precise representation of this bacterial community may be affected by the protocols used for DNA extraction as well as by the PCR primers employed in the amplification reaction. Here, we describe an optimized protocol for 16S rRNA gene-based profiling of the fecal microbiota.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Biodiversity
  • Feces / microbiology*
  • Female
  • Humans
  • Microbiota*
  • RNA, Ribosomal, 16S / chemistry
  • RNA, Ribosomal, 16S / genetics*
  • Rats
  • Sequence Analysis, DNA / methods*

Substances

  • RNA, Ribosomal, 16S

Associated data

  • SRA/SRS490505
  • SRA/SRS490506
  • SRA/SRS490507
  • SRA/SRS490508
  • SRA/SRS490509
  • SRA/SRS490510

Grant support

The authors thank GenProbio srl for the financial support of the Laboratory of Probiogenomics. This work was financially supported by Fondazione Cariparma to MV and by a FEMS Advanced Fellowship 2011 and an IRCSET Embark postdoctoral fellowship to FT. This work was also financially supported by a Ph.D. fellowship (Spinner 2013, Regione Emilia Romagna) to S.D. DvS is a member of The Alimentary Pharmabiotic Centre, which is a Centre for Science and Technology (CSET) funded by the Science Foundation Ireland (SFI), through the Irish Government’s National Development Plan (Grant no. 02/CE/B124 and 07/CE/B1368). The authors also acknowledge the support of the Spanish Plan Nacional de I+D (grant AGL2010-14952). Arancha Hevia was the recipient of an FPI grant, and Borja Sánchez was the recipient of a Juan de la Cierva postdoctoral contract. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.