DNA reference reagents isolate biases in microbiome profiling: a global multi-lab study

When profiling the human gut microbiome, technical biases introduced by analytical approaches impede translational research, reducing data reliability and study comparability. Here, through a global study involving 23 labs, we analyzed a wide range of sequencing and bioinformatic approaches for the taxonomic profiling of two well-defined DNA reference reagents (RRs) comprised of 20 common gut bacteria. Through both shotgun and 16S rRNA gene amplicon sequencing, we aimed to isolate sources of bias and understand their impact on microbiome profiling accuracy. Importantly, minimum quality criteria (MQC) were established and are used to evaluate profiling performance. We found that the variability of shotgun sequencing data sets was greater than that of 16S rRNA gene amplicon sequencing and isolated sources of bias in wet and dry lab steps, such as sequencing depth, primer and database choices, rarefaction, and 16S copy number adjustment. This study presents well-defined RRs and MQC to combat technical bias, paving the way for reliable and comparable microbiome research.IMPORTANCEThis benchmark paper highlights the true level of variability in microbiome data across the world and across sectors, underscoring the critical need for the use of WHO International DNA Gut Reference Reagents (RRs) to elevate the quality of data in microbiome research. This global study is the first of its kind, revealing the reality of the bias in the field, comprehensively testing methodologies used by leading laboratories across the world, but also providing avenues for workflow optimization, to accelerate innovation and translational research and move the field forward.

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Publication Details

Journal
mSystems
Published
2025-10-17
DOI
https://doi.org/10.1128/msystems.00466-25
Citations
4
Primary Topic
Gut microbiota and health
Type
article
Field-Weighted Citation Impact
3.22
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DNA reference reagents isolate biases in microbiome profiling: a global multi-lab study

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DNA reference reagents isolate biases in microbiome profiling: a global multi-lab study

Alex J. La Reau, Aron C. Eklund, Sukyung Kim, Henrik Bjørn Nielsen, Emma Allen‐Vercoe, David Berry, Bela Hausmann, Alexander Mahnert, Akın Yiğin, Gregory C. A. Amos, Mehmet Fatih Demirci, Samuel P. Forry, Celina Whalley, Sudarshan Anand Shetty, Andrea Telatin, Gary P. Schroth, Irina Khrebtukova, Eugenia Silva‐Herzog, Jasmin Schwarz, David Baker, Julian Roberto Marchesi, Martin Fritzsche, Tonya Ward, Stephanie L. Servetas, Christopher Quince, F W Hyde, Hyun‐Dong Chang, René Riedel, Christine Moissl‐Eichinger, Namita Damle, Christopher Mozsary, Ravneet K. Bhuller, Keith L. Olson, Lizbeth Sayavedra, Robert Kraaij, Niels van Best, Jannike Lea Krause, Arjan Narbad, Rob S. James, Ryan Mate, Lily Laws, Andrew D. Beggs, Susana Fuentes, Sunwha Park, Eric Alden Smith, Sjoerd Rijpkema, Jason G. Kralj, Angelika Kritz, Mila Jhamai, Anoecim Robecca Geelen, John Penders, Scott Kuersten, Sarah J. Vancuren, Wiep Klaas Smits, Nikolaj Sørensen, Georgina L. Hold, Jesús Miguéns Blanco, Jacob Dehinsilu, Xavier Soberón, Scott A. Jackson, Pascal Arp, Angélique B. van ’t Wout, Sébastien Raguideau, Romy D. Zwittink, Matthew T. F. Lamaudière, Andrew Cross, Jonathan Foox, Peter Lenz, Quinten R. Ducarmon, Ed J. Kuijper, Jack Hassall, Joost Verlouw, Chrysi Sergaki, Young Seop Kwon, Monique E. Hunter, Stefano Romano, Hoonhee Seo, R. Anthony Williamson, Daniel Butler, Xabier Vázquez-Campos, Christopher E. Mason, Jennifer N. Dootz, Asad Ul-Haq, Christian A. Sanchez, Nathan Danckert, Birgit Gallé, Richard Stevens, Gudrun Kainz, Giang Truong Le, Gudrun Köhl, S.-Y. Park, Christel Driessen, Mingsheng Guo, Jolanda Kool, Martin Gordon, Shabana Muhammad Anwar, Nynne Nielsen, Ho-Yeon Song, Petra Pjevac, Marcus Rauch, Stephan Rosecker, Aidan Resh, Amanda Busswitz, Shane Sontag
article en
4 citations

Abstract

When profiling the human gut microbiome, technical biases introduced by analytical approaches impede translational research, reducing data reliability and study comparability. Here, through a global study involving 23 labs, we analyzed a wide range of sequencing and bioinformatic approaches for the taxonomic profiling of two well-defined DNA reference reagents (RRs) comprised of 20 common gut bacteria. Through both shotgun and 16S rRNA gene amplicon sequencing, we aimed to isolate sources of bias and understand their impact on microbiome profiling accuracy. Importantly, minimum quality criteria (MQC) were established and are used to evaluate profiling performance. We found that the variability of shotgun sequencing data sets was greater than that of 16S rRNA gene amplicon sequencing and isolated sources of bias in wet and dry lab steps, such as sequencing depth, primer and database choices, rarefaction, and 16S copy number adjustment. This study presents well-defined RRs and MQC to combat technical bias, paving the way for reliable and comparable microbiome research.IMPORTANCEThis benchmark paper highlights the true level of variability in microbiome data across the world and across sectors, underscoring the critical need for the use of WHO International DNA Gut Reference Reagents (RRs) to elevate the quality of data in microbiome research. This global study is the first of its kind, revealing the reality of the bias in the field, comprehensively testing methodologies used by leading laboratories across the world, but also providing avenues for workflow optimization, to accelerate innovation and translational research and move the field forward.

mSystemsVol. 10(11)
University of Vienna (AT), National Institute of Standards and Technology (US), Universität Hamburg (DE), Illumina (United States) (US), Medical University of Graz (AT), Cornell University (US), Soonchunhyang University (KR), Leiden University Medical Center (NL), Maastricht University Medical Centre (NL), Erasmus MC (NL), Quadram Institute (GB), Leibniz Association (DE), UNSW Sydney (AU), Maastricht University (NL), Clinical Microbiomics (Denmark) (DK), Dongwha Pharm (South Korea) (KR), German Rheumatism Research Centre (DE), National Institute for Public Health and the Environment (NL), University Medical Center Hamburg-Eppendorf (DE), National Institute of Genomic Medicine (MX), Diversitech (United States) (US), BioTechMed-Graz (AT), Czech Academy of Sciences, Institute of Microbiology (CZ), Nongwoo Bio (South Korea) (KR), St George Hospital (AU), National Institute of Standards (EG), Source BioScience (United Kingdom) (GB), Cancer Research UK Clinical Trials Unit (GB), Institute of Medical Microbiology and Hygiene (DE), Instituto de Biotecnología de León (ES), Bioinstitut (CZ), St. George Hospital (IN), Instituto de Medicina Genómica (ES), Alphabio (FR), Weill Cornell Medicine (US), Technische Universität Berlin (DE), Imperial College London (GB), Medical University of Vienna (AT), University of Birmingham (GB), University of Guelph (CA), Harran University (TR), Erasmus University Rotterdam (NL)
Openalex Percentile: Top 7%
Gut microbiota and health
3.22
DOIOpen Access PDFOpenAlex
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