Diatom DNA Metabarcoding Passes Its First Cross-Laboratory Stress Test Across Europe

Freshwater ecosystems are under relentless pressure from pollution, nutrient enrichment and habitat degradation, and the microscopic algae known as diatoms have long served as one of the most sensitive early-warning systems for tracking that damage. For decades, assessing the health of rivers and lakes meant training specialists to identify diatom species under a microscope, a slow and labour-intensive process. DNA metabarcoding has promised to change that by reading the genetic signatures of diatom communities directly from environmental samples, and several countries have already trialled the approach at national scale. Yet a stubborn obstacle has remained: every laboratory tends to run the procedure slightly differently, and results from one country or facility cannot always be compared with those from another.

A new study published in the journal Metabarcoding and Metagenomics tackles this problem head-on with one of the most ambitious interlaboratory exercises yet attempted for diatom metabarcoding. Under the coordination of a reference laboratory, 17 participants from 14 countries carried out DNA extraction and PCR amplification in parallel on a shared set of standardised samples, following both a fixed common protocol and their own preferred in-house methods. The goal was to measure precisely where variability creeps into the workflow and whether the ecological conclusions drawn from the data survive the journey between laboratories.

The stakes are higher than they might appear. Environmental legislation across Europe, including the Water Framework Directive, requires member states to classify the ecological status of water bodies using consistent methods. If one country’s DNA-based diatom index systematically produces different scores than another’s, regulators cannot pool results, benchmark progress or trust cross-border comparisons. The research team, led by Valentin Vasselon and colleagues, therefore designed their proficiency test around the two steps of the metabarcoding pipeline that are most notorious for introducing laboratory-specific bias: extracting DNA from biofilm samples and amplifying target genetic markers with the polymerase chain reaction.

Participants received an identical package of test materials, including DNA extracts and biofilm samples collected from rivers and lakes, together with an artificial mock community of known diatom composition. The mock community served as a critical control: because its true composition was known in advance, any deviation in the sequencing results could be traced directly to errors introduced during laboratory processing rather than to genuine ecological differences between samples. Each laboratory performed the work under both conditions, first adhering strictly to a prescribed reference protocol and then applying whatever extraction kit and PCR setup they would normally use in routine monitoring.

The central finding is strikingly positive. When laboratories followed the shared protocol, their results proved highly reproducible, demonstrating that the method can be transferred successfully between facilities in different countries without specialised equipment or bespoke reagents. Even more reassuring for the future of routine monitoring, the ecological assessment outcomes remained highly similar and consistent regardless of which protocol each participant chose to apply. In practical terms, this means that the biological quality scores derived from diatom metabarcoding were robust to the reasonable diversity of laboratory practices currently in use across the scientific community.

That robustness matters because the alternative, forcing every laboratory onto a single rigid protocol, would be both politically and practically difficult. Different countries have invested in different extraction chemistries, primer sets and sequencing platforms over years of method development, and a blanket mandate would discard that accumulated expertise. The study’s authors instead propose a strategy they describe as all for one but prove them all: laboratories may continue using their preferred protocols, provided each one demonstrates its consistency by meeting minimum standard requirements verified through proficiency testing. This reframes standardisation not as uniformity of method but as uniformity of demonstrated performance.

The experimental design also illuminated the specific sources of variability that future standardisation efforts must control. By comparing results across the paired experiments, the team could separate systematic errors, which bias results in a consistent direction, from random errors, which add noise unpredictably. The inclusion of river, lake and mock community samples allowed them to test whether certain sample types or taxonomic compositions were more vulnerable to laboratory effects than others. Such distinctions are essential for deciding which steps of the workflow genuinely need harmonisation and which can safely be left to local preference.

The study arrives at a moment when DNA-based biomonitoring is transitioning from research demonstration to regulatory deployment. National programmes in several European countries have already shown that diatom metabarcoding can reproduce the ecological classifications traditionally obtained by microscopic identification, and in some cases the genetic approach detects additional species and provides finer resolution. What has been missing is the layer of quality assurance that mature analytical disciplines take for granted: ring trials, certified reference materials and documented evidence that a result from one accredited laboratory means the same thing as a result from another. This proficiency test provides a template for building exactly that layer.

The collaborative scale of the exercise is itself noteworthy. Seventeen laboratories spanning 14 countries, from established national reference facilities to smaller academic groups, executed identical experimental workflows in parallel, generating a dataset rich enough to disentangle protocol effects from laboratory effects and sample effects. The author list reflects a genuinely pan-European network of diatom ecologists, molecular biologists and water managers, suggesting that the community has reached the critical mass needed to sustain long-term intercalibration exercises of this kind.

For water managers and regulators, the practical message is that diatom DNA metabarcoding is now technically ready for routine application, provided that laboratories participate in ongoing proficiency testing and demonstrate that their chosen protocols meet agreed minimum standards. For the research community, the study offers a roadmap: standardised sample sets, mock communities as benchmarks, paired protocol comparisons and a reference laboratory to anchor the exercise. If future rounds of testing confirm these findings across more laboratories and more diverse freshwater types, the microscopic algae that have quietly recorded the health of Europe’s rivers for over a century may soon have their status read not through a microscope lens but through the language of DNA, with results that laboratories across the continent can finally compare on equal terms.

Subject of Research: Cross-laboratory proficiency testing to standardise diatom DNA metabarcoding for freshwater biomonitoring

Article Title: Proficiency testing and cross-laboratory method comparison to support standardisation of diatom DNA metabarcoding for freshwater biomonitoring

Article References: Vasselon, V., Rivera, S. F., Ács, É., Almeida, S. F. P., Andree, K. B., Apothéloz-Perret-Gentil, L., Bailet, B., Baričević, A., Beentjes, K. K., Bettig, J., Bouchez, A., Capelli, C., Chardon, C., Duleba, M., Elersek, T., Genthon, C., Jablonska, M., Jacas, L., Kahlert, M., … Paix, B. (2025). Proficiency testing and cross-laboratory method comparison to support standardisation of diatom DNA metabarcoding for freshwater biomonitoring. Metabarcoding and Metagenomics, 9, Article e133264. https://doi.org/10.3897/mbmg.9.133264

Image Credits: AI Generated

DOI: 10.3897/mbmg.9.133264

Keywords: diatoms, DNA metabarcoding, biomonitoring, freshwater, proficiency testing, standardisation, PCR amplification, DNA extraction, water quality, ecological assessment, interlaboratory comparison, mock community