Nanopore duplex sequencing as an alternative to Illumina MiSeq sequencing for eDNA-based biomonitoring of coastal aquaculture impacts

Abstract

Oxford Nanopore Technologies has recently launched a duplex sequencing strategy and announced an improved error rate which is in a similar order of magnitude as Illumina sequencing. We therefore conducted a pilot study to assess whether Nanopore duplex sequencing has potential to be used as a technology in eDNA-based marine biomonitoring. Specifically, we investigated bacterial communities of sediment samples collected from Atlantic salmon (Salmo salar) aquaculture installations and compared the ecological trends obtained from short Illumina (V3-V4 region of the 16S rRNA gene) and long Nanopore (full length 16S rRNA gene) sequence reads. The obtained duplex rate of Nanopore amplicon reads with a Phred score ≥ 30 was 36%, notably higher compared to previous reports from bacterial genome sequencing. When inferring alpha- and beta-diversity from Illumina ASVs and Nanopore OTUs, we found highly congruent ecological patterns. Only when collating ASVs and OTUs across taxonomic ranks, beta-diversity analyses of Illumina-data slightly changed, due to the difficulties to assign a taxonomy to short sequence reads. While on family rank, both sequence datasets had good agreement, genus-assignments of Illumina data were critical, resulting in higher disagreement between the two protocols. Our data provide evidence that eDNA-based monitoring of aquaculture-related environmental impacts could equally well be conducted with the improved Nanopore duplex sequencing. We discuss to what extent eDNA-based biomonitoring could benefit from long-read information.