Reference databases, primer choice, and assay sensitivity for environmental metabarcoding: Lessons learnt from a re-evaluation of an eDNA fish assessment in the Volga headwaters

Biodiversity monitoring via environmental DNA, particularly metabarcoding, is evolving into a powerful assessment tool for riverine systems. However, for metabarcoding to be fully integrated into standardized monitoring programmes, some current challenges concerning sampling design, laboratory workflow, and data analysis need to be overcome. Here, we review some of these major challenges and potential solutions. We further illustrate three potential pitfalls, namely the choice of suitable metabarcoding primers, the necessity of complete reference databases, and varying assay sensitivities, by a reappraisal of our-own recently carried out metabarcoding study in the Volga headwaters. TaqMan qPCRs had detected catfish (Silurus glanis) and European eel (Anguilla anguilla), whereas metabarcoding had not, in the same samples. Furthermore, after extending the genetic reference database by 12 additional species and re-analysing the metabarcoding data, we additionally detected the Siberian spiny loach (Cobitis sibirica) and Ukrainian brook lamprey (Eudontomyzon mariae) and reassigned the operational taxonomic units previously assigned to Misgurnus fossilis to Cobitis sibirica. In silico analysis of metabarcoding primer efficiencies revealed considerable variability among primer pairs and among target species, which could lead to strong primer bias and potential false-negatives in metabarcoding studies if not properly compensated for. These results highlight some of the pitfalls of eDNA-metabarcoding as a means of monitoring fish biodiversity in large rivers, which need to be considered in order to fully unleash the full potential of these approaches for freshwater biodiversity monitoring.