Comparative analysis of eDNA metabarcoding and eDNA metagenomics for fish biodiversity estimates using standard and novel filtration methods

Molecular techniques involving environmental DNA (eDNA) are increasingly used for aquatic species detection. Metabarcoding, a widely adopted technique, suffers from primer bias: uneven amplification of species due to primer mismatches. Primer bias can be eliminated by sequencing all eDNA in a sample directly. This method, known as (PCR-free) metagenomics, offers potential benefits for relative abundance estimates, but is seldom applied to fish communities and eDNA. This study used an expanded two-by-two design to compare fish species detection between multi-marker Oxford Nanopore Technologies’ (ONT) metabarcoding and ONT metagenomics (native sequencing) using two filter types (conventional versus coarse). Moreover, we explored methylation patterns obtained from ONT metagenomics. Environmental DNA was collected in a controlled setup and two field settings, one representing a capture-release scenario. All species present in the controlled environment were detected by both metabarcoding and metagenomics. In field settings, metagenomics detected more species than metabarcoding. Coarse filters recovered more species across all sequencing datasets, except in metabarcoding of field settings. Relative read counts between metabarcoding and metagenomics illustrated that primer bias was present in the used primer sets. Most fish metagenomic sequences were identified as European sturgeon ( Acipenser sturio ) across all eDNA samples. We observed three base modifications on the 18S region of A. sturio , where three nucleotide positions showed different methylation patterns between eDNA samples. Our results demonstrate that metabarcoding and metagenomics function complementarily in biodiversity estimates with metagenomics providing additional insights into base modifications. Furthermore, coarse filters offer strong potential for improved species detection in various environments.