Vasselon Valentin, Rivera Sinziana F, Ács Éva, Almeida Salomé Baja, Andree Karl B, Apothéloz-Perret-Gentil Laure, Bailet Bonnie, Baričević Ana, Beentjes Kevin K, Bettig Juliane, Bouchez Agnès, Capelli Camilla, Chardon Cécile, Duleba Mónika, Elersek Tina, Genthon Clémence, Jablonska Maša, Jacas Louis, Kahlert Maria, Kelly Martyn G, Macher Jan-Niklas, Mauri Federica, Moletta-Denat Marina, Mortágua Andreia, Pawlowski Jan, Pérez-Burillo Javier, Pfannkuchen Martin, Pilgrim Erik, Pissaridou Panayiota, Rimet Frédéric, Stanic Karmen, Tapolczai Kálmán, Theroux Susanna, Trobajo Rosa, Van der Hoorn Berry, Vasquez Marlen I, Vidal Marie, Wanless David, Warren Jonathan, Zimmermann Jonas, Paix Benoît
Scimabio-Interface, Thonon-Les-Bains, France.
Swiss Federal Research Institute WSL Agroscope, Birmensdorf, Switzerland.
Metabarcoding Metagenom. 2025 Jan 10;3:1-23. doi: 10.3897/mbmg.9.133264.
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.
底栖硅藻的DNA宏条形码技术已成功应用于国家层面的生物监测，目前在技术上已可考虑用于常规应用。然而，各国之间以及国内的方案和方法仍存在差异，这限制了它们的可转移性以及结果的可比性。为了克服这一问题，将DNA宏条形码技术常规用于硅藻生物监测需要了解该程序不同步骤所引入的变异性来源。在此，我们研究了常规程序的各个要素如何导致欧洲各实验室之间的变异性。我们进行了一组四项实验，重点关注DNA提取和PCR扩增步骤，以评估它们在不同实验室之间的可重复性以及科学界目前应用的不同方案所引入的变异性。在一个参考实验室的指导下，来自14个国家的17名参与者使用相同的固定方案和各自选择的方案并行进行DNA提取和PCR扩增。每位参与者对一组标准化的DNA和生物膜样本（河流、湖泊和模拟群落）进行实验，以调查潜在的系统误差和随机误差。我们的结果显示了一种方案在各实验室之间的成功可转移性，并且无论每位参与者使用何种方案，都能获得高度相似且一致的生态评估结果。我们提出了一种“人人为我，但人人验证”的策略，表明科学界可以使用不同的方案，只要遵循最低标准要求证明其一致性即可。
