DNA reference reagents isolate biases in microbiome profiling: a global multi-lab study
Anwar, Saba; Lamaudiere, Matthew; Hassall, Jack; Dehinsilu, Jacob; Bhuller, Ravneet K; Hold, Georgina L; Vázquez-Campos, Xabier; Mahnert, Alexander; Moissl-Eichinger, Christine; Gallé, Birgit; Kainz, Gudrun; Pjevac, Petra; Hausmann, Bela; Schwarz, Jasmin; Kohl, Gudrun; Berry, David; Vancuren, Sarah J; Allen-Vercoe, Emma; Nielsen, Nynne; Sørensen, Nikolaj; Eklund, Aron; Nielsen, Henrik Bjørn; Riedel, René; Krause, Jannike Lea; Chang, Hyun-Dong; Park, Suenie; Song, Ho-Yeon; Seo, Hoonhee; Ul-Haq, Asad; Kim, Sukyung; Kwon, Yongbin; Park, Sunwha; Soberon, Xavier; Silva-Herzog, Eugenia; Verlouw, Joost AM; Arp, Pascal; Jhamai, Mila; Kraaij, Robert; Geelen, Anoecim R; Ducarmon, Quinten R; Smits, Wiep Klaas; Kuijper, Ed J; Zwittink, Romy D; van Best, Niels; Penders, John; Le, Giang; Driessen, Christel; Kool, Jolanda; Shetty, Sudarshan A; Fuentes, Susana; Demirci, Mehmet; Yigin, Akin; Whalley, Celina; Beggs, Andrew D; Quince, Christopher; James, Rob; Raguideau, Sebastien; Gordon, Martin; Mate, Ryan; Fritzsche, Martin; Danckert, Nathan P; Blanco, Jesus Miguens; Marchesi, Julian R; Rauch, Marcus; Williamson, R Anthony; Van't Wout, Angélique B; Kritz, Angelika; Rosecker, Stephan; Stevens, Richard; Laws, Lynette; Sayavedra, Lizbeth; Romano, Stefano; Telatin, Andrea; Baker, David; Narbad, Arjan; Servetas, Stephanie L; Kralj, Jason G; Forry, Samuel P; Hunter, Monique E; Dootz, Jennifer N; Jackson, Scott A; Mason, Christopher E; Butler, Daniel J; Mozsary, Christopher; Foox, Jonathan; Damle, Namita; Resh, Aidan; Busswitz, Amanda; Lenz, Peter; Sontag, Shane; Cross, Andrew; Sanchez, Christian; Guo, Mingsheng; Olson, Kayla; Smith, Eric A; La Reau, Alex J; Ward, Tonya; Kuersten, Scott; Hyde, Fred; Khrebtukova, Irina; Schroth, Gary; Rijpkema, Sjoerd; Amos, Gregory CA; Sergaki, Chrysi
Series / Report no.
Open Access
Type
Journal Article
Article
Article
Language
en
Date
2025-10-17
Research Projects
Organizational Units
Journal Issue
Title
DNA reference reagents isolate biases in microbiome profiling: a global multi-lab study
Translated Title
Published in
mSystems 2025; 10(11):e0046625
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.