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Person working with an isolation chamber for the study of intestinal microbiota in a confined and controlled environment. © Bertrand NICOLAS, INRA, NICOLAS Bertrand

Human intestinal microbiota: progress towards a standardised protocol for the processing of stool samples

Obtaining extensive knowledge about human intestinal microbiota and assessing its impact on health requires a standardisation of the metagenomic* methods used. As part of a vast international project, researchers from INRA and the CEA1 have revealed the major impact of DNA extraction on the evaluation of the microbial composition of human stool samples. They propose an optimised protocol as part of a series of standardised procedures for processing samples, from their collection to the bioinformatic analysis of the data. Highly efficient, transferable and automatable, this protocol will contribute to the production of quality data which permits robust comparisons to be made. Their results are published in the 02 October 2017 edition of Nature Biotechnology.

Updated on 10/10/2017
Published on 10/05/2017

From the mouth to the anus, the gastrointestinal system is one of the most studied ecosystems. Knowledge of its microbial composition is obtained by extracting and sequencing the DNA of the micro-organisms which inhabit it.  Between collecting the samples and computer analysis, researchers from INRA and the CEA1 and their partners studied the impact of DNA extraction methods on the microbial composition of human stool samples. They developed a standardised DNA extraction protocol to facilitate large-scale analysis of microbiota and the comparison of results.

As part of an international project**, no fewer than 21 DNA extraction protocols have been compared in 11 countries of the northern hemisphere and on three continents (Europe, America and Asia). Commercial extraction kits or actual laboratory protocols were implemented by each partner involved to evaluate the same stool samples. Eight samples in total were collected from two individuals.

DNA extraction: a key step in analysis

Scientists from INRA and the CEA and their partners have demonstrated  that while all steps of sample analysis are critical, DNA extraction is most likely to affect the results, generating greater variations than other biological factors (e.g. differences between individuals or differences over time for a single person) or other technical steps (e.g. how the DNA is conserved).
The quantity of DNA extracted and its integrity are thus broadly affected by the extraction protocol and influence the composition of the sample. Out of 366 bacterial species tested, 90 (25%) are affected by the extraction protocol and may be under-represented. The majority of these are Gram-positive bacteria (which account for some 37 % of microbiote bacteria), the walls of which are more resistant to the mechanical forces mobilised during extraction.

Progress towards a standardised, efficient and transferable protocol

Researchers then assessed the transferability and quantified the accuracy of the most efficient DNA extraction protocols by working on a synthetic microbial community composed of strains representing 10 bacterial species.

After completing these different tests, INRA and CEA scientists and their partners proposed a standardised protocol for the processing of human stool samples, from collecting the samples and bioinformatic analysis to DNA extraction and sequencing.

The international scientific community is investing massively on this subject in the absence of standardised protocols; the researchers in this study, by structuring metagenomic methods, have developed an efficient, transferable and automatable protocol for DNA extraction. The protocol is included in a complete chain of standardised procedures available on-line (http://www.microbiome-standards.org/#), which, in the fields of human, animal and other types of microbiota, will help improve the quality of produced data and its comparison.
 
*Metagenomics consists of collectively studying the genes of a microbial community as a whole (bacteria, viruses, fungi, archaea) without requiring access to laboratory-isolated microbes. The diversity and composition of complex communities is analysed and modelled by high-throughput DNA sequencing.
** This work was carried out as part of the International Human Microbiome Standards project (EU-IHMS, FP7, 2011-2015).

1. Institut François Jacob/Génoscope

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Reference

Towards standards for human fecal sample processing in metagenomic studies

Paul I Costea, Georg Zeller, Shinichi Sunagawa , Eric Pelletier, Adriana Alberti , Florence Levenez, Melanie Tramontano, Marja Driessen, Rajna Hercog, Ferris-Elias Jung, Jens Roat Kultima, Matthew R Hayward, Luis Pedro Coelho, Emma Allen-Vercoe, Laurie Bertrand, Michael Blaut, Jillian R M Brown, Thomas Carton, Stéphanie Cools-Portier, Michelle Daigneault, Muriel Derrien, Anne Druesne, Willem M de Vos, B Brett Finlay, Harry J Flint et al. (2017). Nature Biotechnology. 2 October 2017. DOI:10.1038/nbt.3960