Proficiency testing and cross-laboratory method comparison to support standardisation of diatom DNA metabarcoding for freshwater biomonitoring
Valentin Vasselon, Sinziana F. Rivera, Éva Ács, Salomé F.P. Almeida, Karl B. Andrée, Laure Apothéloz‐Perret‐Gentil, Bonnie Bailet, Ana Baričević, Kevin K. Beentjes, Juliane Bettig, Agnès Bouchez, Camilla Capelli, Cécile Chardon, Mónika Duleba, Tina Eleršek, Clémence Genthon, Maša Jablonska, Louis Jacas, Maria Kahlert, Martyn Kelly, Jan‐Niklas Macher, Federica Mauri, Marina Moletta‐Denat, Andreia Mortágua, Jan Pawłowski, Javier Pérez‐Burillo, Martin Pfannkuchen, Erik M. Pilgrim, Panayiota Pissaridou, Frédéric Rimet, Karmen Stanič, Kálmán Tapolczai, Susanna Theroux, Rosa Trobajo, Berry van der Hoorn, Marlen I. Vasquez, Marie Vidal, David Wanless, Jonathan Warren, Jonas Zimmermann, Benoît Paix
Abstract
DNA metabarcoding of benthic diatoms has been successfully applied for biomonitoring at the national scale and can now be considered technically ready for routine application. However, protocols and methods still vary between and within countries, limiting their transferability and the comparability of results. In order to overcome this, routine use of DNA metabarcoding for diatom biomonitoring requires knowledge of the sources of variability introduced by the different steps of the procedure. Here, we examine how elements of routine procedures contribute to variability between European laboratories. A set of four experiments were performed focusing on DNA extraction and PCR amplification steps to evaluate their reproducibility between different laboratories and the variability introduced by different protocols currently applied by the scientific community. Under the guidance of a reference laboratory, 17 participants from 14 countries performed DNA extraction and PCR amplification in parallel, using the same fixed protocol and their own choice of protocol. Experiments were performed by each participant on a set of standardised DNA and biofilm samples (river, lake and mock community) to investigate potential systematic and random errors. Our results revealed the successful transferability of a protocol amongst labs and a highly similar and consistent ecological assessment outcome obtained regardless of the protocols used by each participant. We propose an "all for one but prove them all" strategy, suggesting that distinct protocols can be used within the scientific community, as long as their consistency is be proven by following minimum standard requirements.