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 assessments are 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 500bp, offering limited taxonomic resolution.  We developed a method for long-read eDNA metabarcoding, using Nanopore sequencing of a longer amplicon and present DECONA, a read processing pipeline to enable improved identification of marine vertebrate species. We designed a universal primer pair targeting a 2kb region of fish mitochondrial DNA and compared it to the commonly used MiFish primer pair targeting ~170bp. In silico testing showed that 2kb fragments improved accurate identification of closely related species. Analyzing eDNA from a North Sea aquarium showed that sequences from both primer pairs could be assigned to most species, and 2kb sequence could detect additional sequences to species level. Interestingly, this difference was opposite in eDNA samples from different locations in the North Sea, where not the 2kb but the MiFish primers identified more species in these locations. This study demonstrates the feasibility of using long-read metabarcoding for eDNA vertebrate biodiversity assessments. However, findings also suggests that as longer fragments are less abundant in the environment, but not in aquarium settings, longer fragments may provide a more recent snapshot of the community. Thus, long-read metabarcoding can expand the molecular toolbox for biodiversity assessments by improving species-level identification and may be especially useful when the temporal origin of the eDNA signal is better understood.