Abstract
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.
| Original language | English |
|---|---|
| Pages (from-to) | 1069–1076 |
| Number of pages | 8 |
| Journal | Nature Biotechnology |
| Volume | 35 |
| Early online date | 2 Oct 2017 |
| DOIs | |
| Publication status | Published - 2017 |
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Keywords
- applied microbiology
- metagenomics
Cite this
Towards standards for human fecal sample processing in metagenomic studies. / Costea, Paul I; Zeller, Georg; Sunagawa, Shinichi; Pelletier, Eric; Alberti, Adriana; Levenez, Florence; Tramontano, Melanie; Driessen, Marja; Hercog, Rajna; Jung, Ferris-Elias; Kultima, Jens Roat; Hayward, Matthew R; Coelho, Luis Pedro; Allen-Vercoe, Emma; Bertrand, Laurie; Blaut, Michael; Brown, Jillian R M; Carton, Thomas; Cools-Portier, Stéphanie; Daigneault, Michelle; Derrien, Muriel; Druesne, Anne; de Vos, Willem M; Finlay, B Brett; Flint, Harry J; Guarner, Francisco; Hattori, Masahira; Heilig, Hans; Luna, Ruth Ann; van Hylckama Vlieg, Johan; Junick, Jana; Klymiuk, Ingeborg; Langella, Philippe; Le Chatelier, Emmanuelle; Mai, Volker; Manichanh, Chaysavanh; Martin, Jennifer C; Mery, Clémentine; Morita, Hidetoshi; O'Toole, Paul W; Orvain, Céline; Patil, Kiran Raosaheb; Penders, John; Persson, Søren; Pons, Nicolas; Popova, Milena; Salonen, Anne; Saulnier, Delphine; Scott, Karen P; Singh, Bhagirath; Slezak, Kathleen; Veiga, Patrick; Versalovic, James; Zhao, Liping; Zoetendal, Erwin G; Ehrlich, S Dusko; Dore, Joel; Bork, Peer.
In: Nature Biotechnology, Vol. 35, 2017, p. 1069–1076.Research output: Contribution to journal › Article
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TY - JOUR
T1 - Towards standards for human fecal sample processing in metagenomic studies
AU - Costea, Paul I
AU - Zeller, Georg
AU - Sunagawa, Shinichi
AU - Pelletier, Eric
AU - Alberti, Adriana
AU - Levenez, Florence
AU - Tramontano, Melanie
AU - Driessen, Marja
AU - Hercog, Rajna
AU - Jung, Ferris-Elias
AU - Kultima, Jens Roat
AU - Hayward, Matthew R
AU - Coelho, Luis Pedro
AU - Allen-Vercoe, Emma
AU - Bertrand, Laurie
AU - Blaut, Michael
AU - Brown, Jillian R M
AU - Carton, Thomas
AU - Cools-Portier, Stéphanie
AU - Daigneault, Michelle
AU - Derrien, Muriel
AU - Druesne, Anne
AU - de Vos, Willem M
AU - Finlay, B Brett
AU - Flint, Harry J
AU - Guarner, Francisco
AU - Hattori, Masahira
AU - Heilig, Hans
AU - Luna, Ruth Ann
AU - van Hylckama Vlieg, Johan
AU - Junick, Jana
AU - Klymiuk, Ingeborg
AU - Langella, Philippe
AU - Le Chatelier, Emmanuelle
AU - Mai, Volker
AU - Manichanh, Chaysavanh
AU - Martin, Jennifer C
AU - Mery, Clémentine
AU - Morita, Hidetoshi
AU - O'Toole, Paul W
AU - Orvain, Céline
AU - Patil, Kiran Raosaheb
AU - Penders, John
AU - Persson, Søren
AU - Pons, Nicolas
AU - Popova, Milena
AU - Salonen, Anne
AU - Saulnier, Delphine
AU - Scott, Karen P
AU - Singh, Bhagirath
AU - Slezak, Kathleen
AU - Veiga, Patrick
AU - Versalovic, James
AU - Zhao, Liping
AU - Zoetendal, Erwin G
AU - Ehrlich, S Dusko
AU - Dore, Joel
AU - Bork, Peer
N1 - 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.
PY - 2017
Y1 - 2017
N2 - 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.
AB - 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.
KW - applied microbiology
KW - metagenomics
UR - http://www.nature.com/authors/policies/license.html
U2 - 10.1038/nbt.3960
DO - 10.1038/nbt.3960
M3 - Article
VL - 35
SP - 1069
EP - 1076
JO - Nature Biotechnology
JF - Nature Biotechnology
SN - 1087-0156
ER -