Towards standards for human fecal sample processing in metagenomic studies
Open accessAbstract
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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Bias can be introduced throughout all stages of sample processing, including selection of sample collection protocol, preservative choice, storage temperature, DNA extraction protocol, library preparation, sequencing platform, and bioinformatics analysis pipeline.
“Bias can be introduced throughout all stages of sample processing, including the selection of sample collection protocol, preservative choice, storage temperature, DNA extraction protocol, library preparation, sequencing platform, and bioinformatics analysis pipeline.720–723”
From Microbiota–gut–brain axis and its therapeutic applications in neurodegenerative diseases (2024) · cites this paper as [721]
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- Authors
- Costea, P. I., Zeller, G., Sunagawa, S., Pelletier, E., Alberti, A., Levenez, F., Tramontano, M., Driessen, M., Hercog, R., Jung, F.-E., Kultima, J. R., Hayward, M. R., Coelho, L. P., Allen-Vercoe, E., Bertrand, L., Blaut, M., Brown, J. R. M., Carton, T., Cools-Portier, S., Daigneault, M., Derrien, M., Druesne, A., de Vos, W. M., Finlay, B. B., Flint, H. J., Guarner, F., Hattori, M., Heilig, H., Luna, R. A., van Hylckama Vlieg, J., Junick, J., Klymiuk, I., Langella, P., Le Chatelier, E., Mai, V., Manichanh, C., Martin, J. C., Mery, C., Morita, H., O'Toole, P. W., Orvain, C., Patil, K. R., Penders, J., Persson, S., Pons, N., Popova, M., Salonen, A., Saulnier, D., Scott, K. P., Singh, B., Slezak, K., Veiga, P., Versalovic, J., Zhao, L., Zoetendal, E. G., Ehrlich, S. D., Dore, J., & Bork, P.
- Affiliations
- European Molecular Biology Laboratory (DE)
- ETH Zurich (CH)
- Commissariat à l'Énergie Atomique et aux Énergies Alternatives (FR)
- Institut National de Recherche pour l'Agriculture, l'Alimentation et l'Environnement (FR)
- University College Cork (IE)
- Danone (France) (FR)
- University of British Columbia (CA)
- University of Aberdeen (GB)
- Vall d'Hebron Institut de Recerca (ES)
- Waseda University (JP)
- Wageningen University & Research (NL)
- Texas Children's Hospital (US)
- German Institute of Human Nutrition (DE)
- Medical University of Graz (AT)
- University of Florida Health (US)
- Okayama University (JP)
- Maastricht University Medical Centre (NL)
- Statens Serum Institut (DK)
- University of Helsinki (FI)
- Western University (CA)
- Shanghai Jiao Tong University (CN)
- Guy's Hospital (GB)
- Corresponding author
- Ehrlich, S Dusko
- Dore, Joel
- ORCID
- Costea, Paul I
- Zeller, Georg
- Sunagawa, Shinichi
- Alberti, Adriana
- Tramontano, Melanie
- Hercog, Rajna
- Jung, Ferris-Elias
- Kultima, Jens Roat
- Hayward, Matthew R
- Coelho, Luis Pedro
- Bertrand, Laurie
- Brown, Jillian R M
- Carton, Thomas
- Daigneault, Michelle
- Derrien, Muriel
- de Vos, Willem M
- Finlay, B Brett
- Flint, Harry J
- Guarner, Francisco
- Hattori, Masahira
- Heilig, Hans
- Luna, Ruth Ann
- Klymiuk, Ingeborg
- Langella, Philippe
- Mai, Volker
- Manichanh, Chaysavanh
- Orvain, Céline
- Patil, Kiran Raosaheb
- Penders, John
- Persson, Søren
- Pons, Nicolas
- Salonen, Anne
- Scott, Karen P
- Singh, Bhagirath
- Veiga, Patrick
- Versalovic, James
- Zhao, Liping
- Zoetendal, Erwin G
- Ehrlich, S Dusko
- Dore, Joel
- Venue
- Nature Biotechnology
- Published
- 2017-10-02
- Volume
- 35
- Issue
- 11
- Pages
- 1069-1076
- ISSN
- 0733-222X (ISSN-L)
- 1087-0156
- 1546-1696
- OpenAlex
- W2763540102
- Cited by
- 893
- References
- 38
- Citations by year
- 2026: 120 · 2025: 94 · 2024: 101 · 2023: 95 · 2022: 112 · 2021: 122 · 2020: 108 · 2019: 82 · 2018: 54 · 2017: 5
- FWCI
- 27.80
- Type
- journal article
- Language
- en
- Open access
- bronze
- doi.org
- Locations
- Nature Biotechnology (publishedVersion)
- PubMed (publishedVersion)
- Research Publications (Maastricht University) (acceptedVersion, other-oa)
- MDC Repository (Max-Delbrueck-Center for Molecular Medicine) (acceptedVersion)
- Socio-Environmental Systems Modeling (acceptedVersion)
- HAL (submittedVersion, other-oa)
- QUT ePrints (Queensland University of Technology) (submittedVersion)
- Research Publications (Maastricht University) (submittedVersion, other-oa)
- Data Archiving and Networked Services (DANS) (submittedVersion)
- Funders
- European Commission (261376)
- Agence Nationale de la Recherche (ANR-10-INBS-0009, ANR-11-DPBS-0001)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Gut microbiota and health
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Phylogenetic Studies
- Physical Sciences › Environmental Science › Ecology › Microbial Community Ecology and Physiology
- Keywords
- human gut microbiome, DNA extraction methods, metagenomics, technical variability, microbial community composition, library preparation, sample storage, biological variation, mock communities, protocol standardization, Gram-positive to Gram-negative ratio
- MeSH terms
- Bacteria, DNA, Feces, Chemical Fractionation, Humans, Quality Control, Species Specificity, Computational Biology, Metagenomics
About these checks
Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.
Statistics and arithmetic are recomputed, not judged.
Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.
What counts as a claim ›How we measure confidence ›How we check this ›
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