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 from seawater is becoming a powerful monitoring tool.
However, current approaches are based on genetic target regions of
<500 nucleotides, which offer limited taxonomic resolution.
This study aims to develop and validate a long read nanopore sequencing
method for eDNA that enables improved identification of fish species. We
designed a universal primer pair targeting a 2kb region covering the 12S
and 16S rRNA genes of fish mitochondria. eDNA was amplified and
sequenced using the Oxford Nanopore MiniON. Sequence data was processed
using the new pipeline Decona, and accurate consensus identities of
above 99.9% were retrieved. The primer set efficiency was tested with
eDNA from a 3.000.000 L zoo aquarium with 31 species of bony fish and
elasmobranchs. Over 55% of the species present were identified on
species level and over 75% on genus level. Next, our long read eDNA
metabarcoding approach was applied to North Sea eDNA field samples
collected at ship wreck sites, the Gemini Offshore Wind Farm, the Borkum
Reef Grounds and a bare sand bottom. Here, location specific fish and
vertebrate communities were obtained. Incomplete reference databases
still form a major bottleneck in further developing high resolution long
read metabarcoding. Yet, the method has great potential for rapid and
accurate fish species monitoring in marine field studies.
High resolution species detection: accurate long read eDNA metabarcoding of North Sea fish using Oxford Nanopore sequencing
