High resolution species detection: accurate long read eDNA metabarcoding of North Sea fish using Oxford Nanopore sequencing

Abstract

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.

A comprehensive evaluation of taxonomic classifiers in marine vertebrate eDNA studies

“…One poten al solu on may involve employing longer sequences or even full-length genes in eDNA metabarcoding. Novel metabarcoding approaches targe ng en re gene sequences improve dis nc on among similar species (Deiner et al, 2017, Doorenspleet et al, 2021. Future work will evaluate the impact of longer or full-length sequences on classifier performance and outcomes.…”

Nano‐Strainer: A workflow for the identification of single‐copy nuclear loci for plant systematic studies, using target capture kits and Oxford Nanopore long reads

“…Several tools for automatic handling of Nanopore amplicon data are available. Examples are NanoRTax (however limited to 16S rRNA amplicons only and intended for taxonomic analysis of microbial community samples, Rodriguez‐Perez et al, 2021) or Decona (for processing data from demultiplexing to consensus calculation, Doorenspleet et al, 2021). However, none of these tools can adequately handle paralogs present in amplified loci.…”

PIMENTA: PIpeline for MEtabarcoding through Nanopore Technology used for Authentication

“…We benchmark the performance (i.e., the number and taxonomic level of identified zooplankton species) and runtime of PIMENTA with two other existing pipelines, the previous pipeline published by Voorhuijzen-Harink et al [2] as well as Decona [4]. An overview of the procedures, analysis and databases applied in this study is shown in Figure 1.…”

“…Metabarcoding data analysis, however, is not yet mainstreamed and many different bioinformatic pipelines have become available over recent years [2,3,4,5,6]. As the sequencing error profile of MinION sequencing differs to that of other sequencing devices such as Illumina sequencers [3,7], analysis pipelines developed with data from other platforms in mind may not perform well on MinION data.…”