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

The research, led by Mariachiara Vardeu and Elena Ghiorzi of the University of Padua together with colleagues at the University of Pavia, was conceived around a central weakness in the existing literature: most tick microbiome studies choose a decontamination method by convention rather than by evidence. Ethanol washes, bleach treatments, and commercial DNA-degrading reagents all appear in published workflows, but until now there has been no direct, controlled comparison of how effectively each removes surface-associated bacteria while preserving the tick’s internal microbial community. Without such a benchmark, differences between laboratories could reflect cleaning chemistry as much as genuine biology, undermining efforts to compare microbiome data across studies, regions, or tick populations.

To build that benchmark, the team designed an elegant experimental trick. Rather than relying on the unknown and variable bacteria naturally present on wild-caught ticks, they deliberately contaminated adult Ixodes ricinus ticks and nymphs with a defined bacterial mock community of known composition. This meant that the researchers knew exactly which bacterial sequences represented surface contamination and could track, with precision, how much of that signal survived each cleaning treatment. Any mock-community reads remaining after decontamination were, by definition, residual surface contamination; any reduction in those reads measured the effectiveness of the protocol. It is a simple idea in principle, but one that transforms decontamination from a matter of faith into a measurable quantity.

The contaminated ticks were then subjected to five decontamination regimens: a wash in 70% ethanol, the traditional mainstay of tick microbiome studies; a 1% bleach treatment; DNA/RNA Zap, a commercial reagent that degrades nucleic acids on external surfaces; and two combination protocols in which a lysozyme pre-treatment, designed to weaken bacterial cell walls, was followed by either bleach or DNA/RNA Zap. Mock-inoculated ticks that received no decontamination served as controls. After cleaning, the researchers characterized the microbial communities using Oxford Nanopore full-length 16S rRNA gene sequencing, a platform that reads the entire bacterial barcoding gene rather than short fragments, providing higher taxonomic resolution for identifying which bacteria survived which treatment.

The results were striking in their clarity. Ethanol, the most widely used decontamination agent in the field, consistently showed the weakest effect, leaving substantial mock-community signal intact and suggesting that many published tick microbiome profiles built on ethanol-washed specimens may carry a meaningful contribution from surface bacteria. By contrast, both bleach and DNA/RNA Zap, whether applied alone or preceded by lysozyme, produced near-complete depletion of the mock-community reads. In other words, the two harsher chemical approaches came closest to erasing the experimental contamination signal, while the gentler ethanol wash barely dented it. Perhaps unexpectedly, the enzymatic lysozyme pre-treatment conferred no measurable advantage over bleach or DNA/RNA Zap used on their own, a finding that simplifies protocol design and removes an unnecessary step from future workflows.

Effectiveness at removing contaminants is only half the story, however. An ideal decontamination protocol must also preserve the tick’s genuine internal microbiota, including endosymbionts that play roles in tick nutrition and reproduction, and any pathogens the tick may harbor. To probe this dimension, the team compared sequencing results from tick legs, which are dominated by surface-associated microbes, with leg-free bodies, which contain the internal organs and their resident communities. This anatomical dissection revealed that Brevibacterium, a bacterial genus frequently reported in tick studies, was predominantly surface-associated in this system, raising the possibility that some taxa previously interpreted as tick symbionts may in fact be exoskeletal passengers. Meanwhile, the endosymbiont Candidatus Midichloria, a bacterium closely associated with Ixodes ticks, remained readily detectable in leg-free bodies even after decontamination, indicating that the effective treatments stripped the surface without erasing the tick’s true internal signature.

The preservation of pathogen signals was equally reassuring. In nymphs that had been experimentally infected with Borrelia, the causative agent of Lyme disease, Borrelia-associated sequences remained readily detectable following surface treatment. This matters enormously for disease surveillance, because ticks are routinely screened for Borrelia and other pathogens using molecular methods, and a decontamination protocol aggressive enough to destroy pathogen DNA would compromise those assays. The study’s findings suggest that bleach- and DNA/RNA Zap-based protocols occupy a valuable middle ground: strong enough to eliminate exoskeletal contamination, yet gentle enough to leave internal microbial and pathogen signals intact and quantifiable.

Beyond the head-to-head protocol comparison, the study also explored how decontamination choice shapes community-level diversity analyses, the statistical frameworks ecologists use to compare microbiomes across samples. These analyses suggested treatment-associated differences in the bacterial profiles recovered from the ticks, hinting that laboratories using different cleaning methods may not be sampling quite the same biological reality. The authors are careful to frame these diversity patterns as exploratory rather than definitive, but the implication is clear: decontamination is not a neutral technical detail but an active variable that can shift the apparent composition of a tick’s microbiome, potentially confounding meta-analyses that pool data from studies using different protocols.

The authors are equally candid about the limits of their benchmark. The mock-community approach, while powerful, was tested under controlled laboratory conditions with a defined and relatively simple surface community. Real ticks collected in the field carry more diverse and unevenly distributed microbial loads, and different tick species present different exoskeleton chemistries and sizes that could influence how reagents penetrate and perform. Validation using field-collected ticks, additional tick species, more complex surface-associated microbial communities, and independent laboratory settings will be required before the community can propose broader methodological standardization. The researchers also emphasize that the optimal decontamination strategy may depend on the biological compartment and microbial fraction of interest, meaning that a one-size-fits-all protocol may never exist.

Nevertheless, the study delivers an immediately actionable message for anyone working on tick microbiomes or tick-borne pathogen surveillance. Under the conditions tested, bleach and DNA/RNA Zap produced the strongest reduction in experimentally introduced surface bacteria while preserving dominant tick-associated taxa and pathogen sequences, whereas ethanol, the field’s default choice, performed worst. Lysozyme pre-treatment added nothing measurable. For a research community racing to understand how tick microbiomes influence vector competence and disease transmission across a changing European landscape, this quantitative framework arrives at an opportune moment. By making surface decontamination a benchmarked, comparable step rather than an unexamined habit, the Padua-led team has taken a meaningful step toward microbiome data that can be trusted, compared, and built upon, and toward a clearer picture of what really lives inside one of Europe’s most medically important arthropods.

Subject of Research: Benchmarking surface decontamination protocols for tick microbiome profiling using 16S rRNA sequencing

Article Title: Benchmarking surface decontamination methods for reliable Ixodes ricinus microbiome profiling

Article References: Vardeu, M., Ghiorzi, E., Bernabè, G., Bellato, M., Vezzi, A., Salata, C., Castagliuolo, I., & Lavezzo, E. (2026). Benchmarking surface decontamination methods for reliable Ixodes ricinus microbiome profiling. Parasites & Vectors. https://doi.org/10.1186/s13071-026-07726-1

Image Credits: AI Generated

DOI: 10.1186/s13071-026-07726-1

Keywords: Ixodes ricinus, tick microbiome, surface decontamination, 16S rRNA sequencing, Oxford Nanopore, mock community, Borrelia, Midichloria, Brevibacterium, Lyme disease, vector competence, Parasites & Vectors