Proficiency testing and cross-laboratory method comparison to support standardisation of diatom DNA metabarcoding for freshwater biomonitoring
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.
Authors
- Kálmán Tapolczai (ORCID: https://orcid.org/0000-0003-1453-767X)
- Ana Baričević (ORCID: https://orcid.org/0000-0002-7082-1977)
- Frédéric Rimet (ORCID: https://orcid.org/0000-0002-5514-869X)
- Camilla Capelli (ORCID: https://orcid.org/0000-0002-4617-305X)
- Bonnie Bailet (ORCID: https://orcid.org/0000-0001-8784-5687)
- Karmen Stanič (ORCID: https://orcid.org/0000-0002-1239-0427)
- Clémence Genthon
- Benoît Paix (ORCID: https://orcid.org/0000-0001-8159-8574)
- Laure Apothéloz‐Perret‐Gentil (ORCID: https://orcid.org/0000-0002-8592-3079)
- David Wanless
- Marie Vidal
- Valentin Vasselon (ORCID: https://orcid.org/0000-0001-5038-7918)
- Martyn Kelly (ORCID: https://orcid.org/0000-0002-4582-5001)
- Cécile Chardon
- Susanna Theroux (ORCID: https://orcid.org/0000-0002-9812-7856)
- Louis Jacas
- Andreia Mortágua (ORCID: https://orcid.org/0000-0003-0182-0513)
- Federica Mauri (ORCID: https://orcid.org/0000-0001-8502-8798)
- Kevin K. Beentjes (ORCID: https://orcid.org/0000-0001-7669-3809)
- Salomé F.P. Almeida (ORCID: https://orcid.org/0000-0001-7240-7967)
- Tina Eleršek (ORCID: https://orcid.org/0000-0003-3296-3808)
- Erik M. Pilgrim
- Maria Kahlert (ORCID: https://orcid.org/0000-0001-9643-4281)
- Sinziana F. Rivera (ORCID: https://orcid.org/0000-0002-0812-9031)
- Panayiota Pissaridou (ORCID: https://orcid.org/0000-0003-4841-7676)
- Marlen Ines Vasquez (ORCID: https://orcid.org/0000-0002-9849-5616)
- Marina Moletta‐Denat
- Berry van der Hoorn
- Javier Pérez‐Burillo (ORCID: https://orcid.org/0000-0002-8489-2389)
- Jonas Zimmermann (ORCID: https://orcid.org/0000-0002-0522-0569)
- Maša Jablonska (ORCID: https://orcid.org/0000-0003-1988-3600)
- Mónika Duleba (ORCID: https://orcid.org/0000-0003-2352-8943)
- Karl B. Andrée (ORCID: https://orcid.org/0000-0001-6564-0015)
- Agnès Bouchez (ORCID: https://orcid.org/0000-0001-8802-6966)
- Éva Ács (ORCID: https://orcid.org/0000-0003-1774-157X)
- Juliane Bettig
- Martin Pfannkuchen (ORCID: https://orcid.org/0000-0002-6253-4716)
- Jan W. Pawlowski (ORCID: https://orcid.org/0000-0003-2421-388X)
- Jan‐Niklas Macher (ORCID: https://orcid.org/0000-0003-3010-7522)
- Jonathan Warren (ORCID: https://orcid.org/0000-0003-3381-3852)
- Rosa Trobajo (ORCID: https://orcid.org/0000-0001-9498-3797)
Institutions
- Southern California Coastal Water Research Project (US)
- University of Geneva (CH)
- Naturalis Biodiversity Center (NL)
- Environmental Protection Agency (US)
- Department for Environment Food and Rural Affairs (GB)
- Ludovika University of Public Service (HU)
- University of Nottingham (GB)
- University of Applied Sciences and Arts of Southern Switzerland (CH)
- University of Ljubljana (SI)
- Cyprus University of Technology (CY)
- Environment Agency (GB)
- Durham University (GB)
- Swiss Federal Institute for Forest, Snow and Landscape Research (CH)
- Interface (United States) (US)
- Swedish University of Agricultural Sciences (SE)
- Institut National de Recherche pour l'Agriculture, l'Alimentation et l'Environnement (FR)
- Institute of Agrifood Research and Technology (ES)
- Ospedale Regionale di Mendrisio (CH)
- Laboratoire de Biotechnologie de l'Environnement (FR)
- Electro Medical Systems (Switzerland) (CH)
- Organizzazione Sociopsichiatrica Cantonale (CH)
- INRA Transfert (France) (FR)
- HUN-REN Balaton Limnological Research Institute (HU)
- Ruđer Bošković Institute (HR)
- Agroscope (CH)
- National Institute of Biology (SI)
- University of Aveiro (PT)
- Université Savoie Mont Blanc (FR)
- Freie Universität Berlin (DE)
Publication Details
- Journal
- Metabarcoding and Metagenomics
- Published
- 2025-01-10
- DOI
- https://doi.org/10.3897/mbmg.9.133264
- Citations
- 11
- Primary Topic
- Environmental DNA in Biodiversity Studies
- Type
- article
- Field-Weighted Citation Impact
- 18.88
Funders
- U.S. Environmental Protection Agency
- Cyprus University of Technology
- Havs- och Vattenmyndigheten
- University of Cyprus
- Centres de Recerca de Catalunya
- Biodiversa+
- Javna Agencija za Raziskovalno Dejavnost RS
- Naturvårdsverket
- Hrvatska Zaklada za Znanost
- Office Français de la Biodiversité
- Institut National de Recherche pour l'Agriculture, l'Alimentation et l'Environnement
- Interreg
- Eurostars