eDNAqua-Plan Deliverable 12: Report on workflow pitfalls

Environmental DNA (eDNA) workflows are increasingly important for aquatic biodiversity monitoring, but the steps from sampling and sequencing to analysis and publication remain fragmented and difficult to align with FAIR (Findable, Accessible, Interoperable, Reusable) principles. The EU project eDNAqua-Plan seeks to address this by developing a digital landscape (proposed by Work Package 3) and testing its operational feasibility across the eDNA workflow through the combined expertise of freshwater and marine use cases (Work Package 4). To this end, WP4 focused on 1) developing effective DMPs for aquatic eDNA studies, 2) streamlining the submission of sequence data to ENA as European INSDC repository, 3) enhancing interoperability across different data components of eDNA projects, 4) making laboratory protocols and 5) bioinformatic workflows FAIR, 6) evaluating eDNA data uploads to global biodiversity portals and 7) aiming for reliable taxonomic assignment of sequence data in eDNA studies.The objective of this deliverable is to identify strengths and challenges of the current digital landscape and to provide specific recommendations to make eDNA data flows more efficient, interoperable and FAIR-compliant.The use cases found that current Data Management Plan (DMP) platforms have generic templates, leaving uncertainty about which datasets need to be added, which metadata need to be provided, and which standards need to be used to have a meaningful DMP. Submitting sequence data to ENA (as the central European INSDC eDNA repository) was identified by use cases to be impractical and insufficiently automated, while interoperability across platforms is hampered by misalignment between the recommended FAIRe checklist and ENA (and also NCBI) templates. The structured BeBOP laboratory protocols offer valuable templates for PCR-based protocols that can be tailored to study specific workflows. The metagenomic template is, however, insufficient to properly describe a metagenomics workflow. Similarly, bioinformatic workflows for eDNA metabarcoding datasets are well covered by existing metadata standards, while key processes in metagenomics such as assembly, binning, and annotation are currently poorly documented in the FAIRe checklist. Uploads to biodiversity portals like GBIF and OBIS are facilitated by the Metabarcoding Toolkit, which is a well documented and easy process. Finally, a continuous central challenge in eDNA studies is assigning a robust taxonomic name to eDNA sequences. Even when all metadata of a reference sequence are provided, this does not guarantee a correct taxonomic identification of the specimen from which the sequence was derived.Based on these challenges, WP4 recommends developing eDNA-specific DMP templates that integrate existing standards such as MIxS, DwC, and FAIRe. ENA submissions should be simplified with better integration of FAIRe metadata and short, user-friendly guidelines. Interoperability across repositories should be strengthened by developing conversion scripts to align FAIRe fields with repository templates. To make laboratory protocols FAIR, more accessible controlled vocabularies are needed. The FAIRe checklist, BeBOP laboratory protocols and biodiversity portals should integrate metagenomic extensions to make the digital landscape ready for the future. Finally, reliable taxonomic assignment requires curated reference databases and bioinformatic scripts with quality control steps to minimise wrong taxonomic assignments.In all, even small steps can make a big difference in lowering barriers for users, ensuring that the digital landscape is truly adopted. Therefore, materials that increase the operational aspect of the digital landscape created by WP4 partners during the testing phase are made available to the eDNA community through public repositories.