Assessing the Fecal Microbiota: An Optimized Ion Torrent 16S rRNA Gene-Based Analysis Protocol

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 nove...

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Published inPloS one Vol. 8; no. 7; p. e68739
Main Authors Milani, Christian, Hevia, Arancha, Foroni, Elena, Duranti, Sabrina, Turroni, Francesca, Lugli, Gabriele Andrea, Sanchez, Borja, Martín, Rebeca, Gueimonde, Miguel, van Sinderen, Douwe, Margolles, Abelardo, Ventura, Marco
Format Journal Article
LanguageEnglish
Published United States Public Library of Science 15.07.2013
Public Library of Science (PLoS)
Subjects
Analysis
Animals
Bacteria
Biodiversity
Bioinformatics
Biological samples
Biology
Deoxyribonucleic acid
DNA
DNA sequencing
Fecal microflora
Feces
Feces - microbiology
Female
Gastrointestinal diseases
Gene sequencing
Genes
Health care
Humans
Intestinal microflora
Laboratories
Life Sciences
Microbiota
Microbiota (Symbiotic organisms)
Microorganisms
Nucleotide sequencing
Phylogenetics
Primers
Rats
RNA
RNA, Ribosomal, 16S - chemistry
RNA, Ribosomal, 16S - genetics
rRNA 16S
Sequence Analysis, DNA - methods
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ISSN1932-6203
1932-6203
DOI10.1371/journal.pone.0068739

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Summary: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.
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Conceived and designed the experiments: MV AM DV MG BS RM. Performed the experiments: CM AH EF SD FT GL. Analyzed the data: MV AM. Contributed reagents/materials/analysis tools: MV AM. Wrote the paper: MV AM DV.
Competing Interests: GenProbio srl provided the financial support of the Laboratory of Probiogenomics for this study. There are no patents, products in development or marketed products to declare. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials, as detailed online in the guide for authors.
ISSN:1932-6203
1932-6203
DOI:10.1371/journal.pone.0068739