The long and the short of it: Nanopore based eDNA metabarcoding of marine vertebrates works; sensitivity and specificity depend on amplicon lengths

To monitor the effect of nature restoration projects in North Sea ecosystems, accurate and intensive biodiversity assessments are vital. DNA based techniques and especially environmental DNA (eDNA) metabarcoding is becoming a powerful monitoring tool. However, current approaches are based on genetic target regions <500 nucleotides, which offer limited taxonomic resolution. We developed a method for eDNA metabarcoding, based on nanopore sequencing of a longer amplicon, enabling improved identification of fish species. We designed a universal primer pair targeting a 2kb region of fish mitochondria, and compared it to the commonly used MiFish primer pair that targets only [~]170bp. In sillico and mock community testing showed that the 2kb fragments improved the accurate identification of genetically closely related species. eDNA was amplified, and sequenced using the Oxford Nanopore MinION in combination with the sequence read processing pipeline Decona. Analyzing eDNA from a North Sea aquarium showed that sequences from both primer pairs could be assigned to most species, but both approaches also identified unique species in the aquarium eDNA. Next, both primer pairs were used on multiple eDNA samples from the North Sea. Here, similar location specific fish communities were obtained from both approaches. More species were identified through the MiFish approach in the field samples. Interestingly, this difference was not observed in the aquarium, suggesting that 2kb fragment based metabarcoding potentially detects more recent occurrences of animals. This new method has the potential to improve and expand the molecular toolbox for eDNA based monitoring approaches.