Proficiency testing and cross-laboratory method comparison to support standardisation of diatom DNA metabarcoding for freshwater biomonitoring
Metabarcoding and Metagenomics, vol. 9, pp. 1–23
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.
Authors 40
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Affiliation as printed
Scimabio-Interface, Thonon-Les-Bains, France
SCIMABIO Interface SAS (5 rue des Quatre Vents, 74200 Thonon-les-Bains - France)
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Swiss Federal Institute for Forest, Snow and Landscape Research
Affiliation as printed
Swiss Federal Research Institute WSL Agroscope, Birmensdorf, Switzerland
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Ludovika University of Public Service
Affiliation as printed
University of Public Service, Baja, Hungary
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Ludovika University of Public Service · University of Aveiro
Affiliation as printed
University of Aveiro, Aveiro, Portugal
Ludovika University of Public Service (Hungary)
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Institute of Agrifood Research and Technology
Affiliation as printed
IRTA, La Ràpita, Spain
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Affiliation as printed
Unaffiliated, Nyon, Switzerland
Agroscope (Berne - Switzerland)
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Institut National de Recherche pour l'Agriculture, l'Alimentation et l'Environnement · Dynamique et durabilité des écosystèmes : de la source à l'océan
Affiliation as printed
UMR DECOD, INRAE, Rennes, France
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Affiliation as printed
Ruder Boskovic Institute, Rovinj, Croatia
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Kevin K. Beentjes Aachen Naturalis Biodiversity Center Naturalis Biodiversity Center [Leiden] (Darwinweg 2
Affiliation as printed
Naturalis Biodiversity Center, Leiden, Netherlands
Naturalis Biodiversity Center [Leiden] (Darwinweg 2, 2333 CR Leiden - Netherlands)
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Affiliation as printed
Freie Universität Berlin, Berlin, Germany
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Affiliation as printed
Université de Savoie Mont-Blanc, Thonon-Les-Bains, France
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University of Applied Sciences and Arts of Southern Switzerland
Affiliation as printed
SUPSI, Institute of Earth Sciences, Mendrisio, Switzerland
SUPSI - Scuola universitaria professionale della Svizzera italiana = University of Applied Sciences and Arts of Southern Switzerland [Manno] (Galleria 2 CH - 6928 Manno Switzerland - Switzerland)
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Affiliation as printed
Université de Savoie Mont-Blanc, Thonon-Les-Bains, France
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Ludovika University of Public Service
Affiliation as printed
University of Public Service, Baja, Hungary
Ludovika University of Public Service (Hungary)
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Affiliation as printed
National Institute of Biology, Ljubljana, Slovenia
NIB - National Institute of Biology [Ljubljana] (Ljubljana - Slovenia)
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Institut National de Recherche pour l'Agriculture, l'Alimentation et l'Environnement · Génome et Transcriptome - Plateforme Génomique
Affiliation as printed
INRAE Transfert - Metys- Service Génomique Plateforme GeT-PlaGe, Castanet-Tolosan, France
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University of Ljubljana · National Institute of Biology
Affiliation as printed
National Institute of Biology, Ljubljana, Slovenia
University of Ljubljana, Ljubljana, Slovenia
NIB - National Institute of Biology [Ljubljana] (Ljubljana - Slovenia)
University of Ljubljana (Kongresni trg 12, 1000 Ljubljana - Slovenia)
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Affiliation as printed
Université de Savoie Mont-Blanc, Thonon-Les-Bains, France
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Swedish University of Agricultural Sciences
Affiliation as printed
Swedish University of Agricultural Sciences, Uppsala, Sweden
SLU - Swedish University of Agricultural Sciences = Sveriges lantbruksuniversitet (S-750 07 Uppsala - Sweden)
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Affiliation as printed
University of Nottingham, Durham, United Kingdom
Bowburn Consultancy (United Kingdom)
UON - University of Nottingham, UK (University of Nottingham University Park Nottingham NG7 2RD - United Kingdom)
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Jan‐Niklas Macher Aachen Naturalis Biodiversity Center Naturalis Biodiversity Center [Leiden] (Darwinweg 2
Affiliation as printed
Naturalis Biodiversity Center, Leiden, Netherlands
Naturalis Biodiversity Center [Leiden] (Darwinweg 2, 2333 CR Leiden - Netherlands)
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University of Applied Sciences and Arts of Southern Switzerland
Affiliation as printed
SUPSI, Institute of Microbiology, Mendrisio, Switzerland
SUPSI - Scuola universitaria professionale della Svizzera italiana = University of Applied Sciences and Arts of Southern Switzerland [Manno] (Galleria 2 CH - 6928 Manno Switzerland - Switzerland)
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Institut National de Recherche pour l'Agriculture, l'Alimentation et l'Environnement · Laboratoire de Biotechnologie de l'Environnement
Affiliation as printed
INRAE Transfert - Metys- LBE- Service Génomique, Narbonne, France
LBE - Laboratoire de Biotechnologie de l'Environnement [Narbonne] (Avenue des Etangs - 11100 Narbonne - France)
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Affiliation as printed
University of Aveiro, Aveiro, Portugal
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Affiliation as printed
University of Geneva, Geneva, Switzerland
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Institute of Agrifood Research and Technology
Affiliation as printed
IRTA, La Ràpita, Spain
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Affiliation as printed
Ruder Boskovic Institute, Rovinj, Croatia
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Environmental Protection Agency
Affiliation as printed
US Environmental Protection Agency, Cincinnati, United States of America
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Cyprus University of Technology
Affiliation as printed
Cyprus University of Technology, Limassol, Cyprus
Cyprus University of Technology (30, Arch. Kyprianos str. CY-3036 Limassol Cyprus - Cyprus)
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Affiliation as printed
Université de Savoie Mont-Blanc, Thonon-Les-Bains, France
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HUN-REN Balaton Limnological Research Institute
Affiliation as printed
HUN-REN Balaton Limnological Research Institute, Tihany, Hungary
Balaton Limnological Research Institute (Klebelsberg Kuno str. 3 PO Box 35 HU-8237 Tihany - Hungary)
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Southern California Coastal Water Research Project
Affiliation as printed
SCCWRP, Costa Mesa, United States of America
SCCWRP - Southern California Coastal Water Research Project (3535 Harbor Blvd., Costa Mesa, CA 92626, USA - United States)
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Institute of Agrifood Research and Technology
Affiliation as printed
IRTA, La Ràpita, Spain
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Berry van der Hoorn Aachen Naturalis Biodiversity Center Naturalis Biodiversity Center [Leiden] (Darwinweg 2
Affiliation as printed
Naturalis Biodiversity Center, Leiden, Netherlands
Naturalis Biodiversity Center [Leiden] (Darwinweg 2, 2333 CR Leiden - Netherlands)
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Cyprus University of Technology
Affiliation as printed
Cyprus University of Technology, Limassol, Cyprus
Cyprus University of Technology (30, Arch. Kyprianos str. CY-3036 Limassol Cyprus - Cyprus)
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Institut National de Recherche pour l'Agriculture, l'Alimentation et l'Environnement · Génome et Transcriptome - Plateforme Génomique
Affiliation as printed
INRAE Transfert - Metys- Service Génomique Plateforme GeT-PlaGe, Castanet-Tolosan, France
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Southern California Coastal Water Research Project
Affiliation as printed
SCCWRP, Costa Mesa, United States of America
SCCWRP - Southern California Coastal Water Research Project (3535 Harbor Blvd., Costa Mesa, CA 92626, USA - United States)
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Department for Environment Food and Rural Affairs · Environment Agency
Affiliation as printed
Environment Agency, Bristol, United Kingdom
DEFRA - Department for Environment, Food & Rural Affairs (London - United Kingdom)
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Affiliation as printed
Freie Universität Berlin, Berlin, Germany
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Affiliation as printed
Université de Savoie Mont-Blanc, Thonon-Les-Bains, France
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