The Long and the Short of It: Nanopore‐Based eDNA Metabarcoding of Marine Vertebrates Works; Sensitivity and Species‐Level Assignment Depend on Amplicon Lengths

To monitor the effect of nature restoration projects in North Sea ecosystems, accurate and intensive biodiversity assessmentsare vital. DNA-based techniques and especially environmental (e)DNA metabarcoding is becoming a powerful monitoring tool.However, current approaches rely on genetic target regions under 500 bp, offering limited taxonomic resolution. We developeda method for long-read eDNA metabarcoding, using Nanopore sequencing of a longer amplicon and present DECONA, a readprocessing pipeline to enable improved identification of marine vertebrate species. We designed a universal primer pair target-ing a 2 kb region of fish mitochondrial DNA and compared it to the commonly used MiFish primer pair targeting a ~ 170 bpregion. In silico testing showed that 2 kb fragments improved accurate identification of closely related species. Analysing eDNAfrom a North Sea aquarium showed that sequences from both primer pairs could be assigned to most species, and additionalspecies level assignments could be made through the 2 kb primer pair. Interestingly, this difference was opposite in eDNA fromthe North Sea, where not the 2 kb but the MiFish primer pair detected more species. This study demonstrates the feasibility ofusing long-read metabarcoding for eDNA vertebrate biodiversity assessments. However, our findings suggests that longer frag-ments are less abundant in environmental settings, but not in aquarium settings, suggesting that longer fragments may providea more recent snapshot of the community. Thus, long-read metabarcoding can expand the molecular toolbox for biodiversityassessments by improving species-level identification and may be especially useful when the temporal origin of the eDNA signalis better understood