Towards standards for human fecal sample processing in metagenomic studies

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Technical variation in metagenomic analysis must be minimized to confidently assess the contributions of microbiota to human health. Here we tested 21 representative DNA extraction protocols on the same fecal samples and quantified differences in observed microbial community composition. We compared them with differences due to library preparation and sample storage, which we contrasted with observed biological variation within the same specimen or within an individual over time. We found that DNA extraction had the largest effect on the outcome of metagenomic analysis. To rank DNA extraction protocols, we considered resulting DNA quantity and quality, and we ascertained biases in estimates of community diversity and the ratio between Gram-positive and Gram-negative bacteria. We recommend a standardized DNA extraction method for human fecal samples, for which transferability across labs was established and which was further benchmarked using a mock community of known composition. Its adoption will improve comparability of human gut microbiome studies and facilitate meta-analyses.

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We thank S. Burz and K. Weizer for editing and web-posting the SOPs. We thank D. Ordonez and N.P. Gabrielli Lopez for advice on flow cytometry, which was provided by the Flow Cytometry Core Facility, EMBL. This study was funded by the European Community's Seventh Framework Programme via International Human Microbiome Standards (HEALTH-F4-2010-261376) grant. We also received support from Scottish Government Rural and Environmental Science and Analytical Services as well as from EMBL.

Author information


  1. Structural and Computational Biology, European Molecular Biology Laboratory, Heidelberg, Germany.

    • Paul I Costea
    • , Georg Zeller
    • , Shinichi Sunagawa
    • , Melanie Tramontano
    • , Marja Driessen
    • , Rajna Hercog
    • , Ferris-Elias Jung
    • , Jens Roat Kultima
    • , Matthew R Hayward
    • , Luis Pedro Coelho
    • , Kiran Raosaheb Patil
    •  & Peer Bork
  2. Department of Biology, Institute of Microbiology, ETH Zurich, Zurich, Switzerland.

    • Shinichi Sunagawa
  3. CEA - Institut François Jacob - Genoscope, Evry, France.

    • Eric Pelletier
    • , Adriana Alberti
    • , Laurie Bertrand
    •  & Céline Orvain
  4. CNRS UMR-8030, Evry, France.

    • Eric Pelletier
  5. Université Evry Val d'Essonne, Evry, France.

    • Eric Pelletier
  6. Metagenopolis, Institut National de la Recherche Agronomique, Jouy en Josas, France.

    • Florence Levenez
    • , Michelle Daigneault
    • , Philippe Langella
    • , Emmanuelle Le Chatelier
    • , Nicolas Pons
    • , S Dusko Ehrlich
    •  & Joel Dore
  7. Department of Molecular and Cellular Biology, The University of Guelph, Guelph, Ontario, Canada.

    • Emma Allen-Vercoe
  8. Department of Gastrointestinal Microbiology, German Institute of Human Nutrition Potsdam-Rehbruecke, Nuthetal, Germany.

    • Michael Blaut
    • , Jana Junick
    • , Delphine Saulnier
    •  & Kathleen Slezak
  9. School of Microbiology & APC Microbiome Institute, University College Cork, Cork, Ireland.

    • Jillian R M Brown
    •  & Paul W O'Toole
  10. Biofortis, Mérieux NutriSciences, Nantes, France.

    • Thomas Carton
    • , Clémentine Mery
    •  & Milena Popova
  11. Danone Nutricia Research, Palaiseau, France.

    • Stéphanie Cools-Portier
    • , Muriel Derrien
    • , Anne Druesne
    • , Johan van Hylckama Vlieg
    •  & Patrick Veiga
  12. Laboratory of Microbiology, Wageningen University & Research, Wageningen, the Netherlands.

    • Willem M de Vos
    • , Hans Heilig
    •  & Erwin G Zoetendal
  13. Immunobiology Research Program, Department of Bacteriology and Immunology, University of Helsinki, Helsinki, Finland.

    • Willem M de Vos
    •  & Anne Salonen
  14. Michael Smith Laboratories, University of British Columbia, Vancouver, British Columbia, Canada.

    • B Brett Finlay
  15. Rowett Institute of Nutrition and Health, University of Aberdeen, Aberdeen, UK.

    • Harry J Flint
    • , Jennifer C Martin
    •  & Karen P Scott
  16. Digestive System Research Unit, Vall d'Hebron Research Institute, Barcelona, Spain.

    • Francisco Guarner
    •  & Chaysavanh Manichanh
  17. Graduate School of Frontier Sciences, The University of Tokyo, Chiba, Japan.

    • Masahira Hattori
  18. Graduate School of Advanced Science and Engineering, Waseda University, Tokyo, Japan.

    • Masahira Hattori
  19. Texas Children's Hospital, Feigin Center, Houston, Texas, USA.

    • Ruth Ann Luna
    •  & James Versalovic
  20. Center for Medical Research, Medical University of Graz, Graz, Austria.

    • Ingeborg Klymiuk
  21. Department of Epidemiology, College of Public Health and Health Professions and College of Medicine, Emerging Pathogens Institute, University of Florida, Gainesville, Florida, USA.

    • Volker Mai
  22. Graduate School of Environmental and Life Science, Okayama University, Okayama, Japan.

    • Hidetoshi Morita
  23. School of Nutrition and Translational Research in Metabolism (NUTRIM) and Care and Public Health Research Institute (Caphri), Department of Medical Microbiology, Maastricht University Medical Center, Maastricht, the Netherlands.

    • John Penders
  24. Unit of Foodborne Infections, Department of Bacteria, Parasites & Fungi, Statens Serum Institut, Copenhagen, Denmark.

    • Søren Persson
  25. Centre for Human Immunology, Department of Microbiology & Immunology and Robarts Research Institute, University of Western Ontario, London, Ontario, Canada.

    • Bhagirath Singh
  26. Ministry of Education Key Laboratory for Systems Biomedicine, Shanghai Centre for Systems Biomedicine, Shanghai Jiao Tong University, Shanghai, PR China.

    • Liping Zhao
  27. King's College London, Centre for Host-Microbiome Interactions, Dental Institute Central Office, Guy's Hospital, London, UK.

    • S Dusko Ehrlich
  28. Department of Bioinformatics, Biocenter, University of Würzburg, Würzburg, Germany.

    • Peer Bork
  29. Molecular Medicine Partnership Unit, Heidelberg, Germany.

    • Peer Bork
  30. Max-Delbrück-Centre for Molecular Medicine, Berlin, Germany.

    • Peer Bork


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P.I.C., S.S. and G.Z. analyzed data and drafted and finalized the manuscript. E.P. and A.A. analyzed data, sequenced samples and wrote the manuscript. F.L., J.R.K., M.R.H., L.P.C. and E.A.-V. analyzed data and wrote the manuscript. M.T., M. Driessen, R.H., F.-E.J. and K.R.P. created and quantified the mock community. M.B., J.R.M.B., L.B., T.C., S.C.-P., M. Derrien, A.D., M. Daigneault, R.A.L., W.M.d.V., B.B.F., H.J.F., F.G., M.H., H.H., J.v.H.V., J.J., I.K., P.L., E.L.C., V.M., C. Manichanh, J.C.M., C. Mery, H.M., C.O., P.W.O., J.P., S.P., N.P., M.P., A.S., D.S., K.P.S., B.S., K.S., P.V., J.V., L.Z. and E.G.Z. extracted samples and wrote the manuscript. S.D.E., J.D. and P.B. designed the study and wrote the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to S Dusko Ehrlich or Joel Dore or Peer Bork.

Integrated supplementary information

Supplementary information

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  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–5

  2. 2.

    Life Sciences Reporting Summary

  3. 3.

    Supplementary Methods

    Supplementary Methods

Excel files

  1. 1.

    Supplementary Data 1

    Protocol descriptors

  2. 2.

    Supplementary Data 2

    Members and composition of mock community

  3. 3.

    Supplementary Data 3

    Sample description