Advancing molecular macrobenthos biodiversity monitoring: a comparison between Oxford Nanopore and Illumina based...

DNA-based methods and developments of sequencing technologies are integral to macrobenthos biodiversity studies, and their implementation as standardized monitoring methods is approaching. Evaluating the efficacy and reliability of these technological developments is crucial for macrobenthos biodiversity assessments. In this study, we compared three DNA-based techniques for assessing the diversity of bulk macrobenthos samples from the Belgian North Sea. Specifically, we compared amplicon sequencing using Illumina MiSeq and portable real-time sequencing of Oxford Nanopore versus shotgun sequencing using Illumina NovaSeq sequencing. The 313 bp mitochondrial cytochrome c oxidase subunit I (COI) metabarcoding fragment served as the target region for the metabarcoding analysis. Our results indicate that Oxford Nanopore and MiSeq metabarcoding had similar performances in terms of alpha and beta diversity, revealing highly similar location-specific community compositions. The NovaSeq metagenomics method also resulted in similar alpha diversity, but slightly different community compositions compared to the metabarcoding approach. Despite these differences, location-specific community compositions were maintained across all platforms. Notably, read counts from the NovaSeq metagenomic analysis showed the weakest correlation to size corrected morphological abundance and there were mismatches between morphological identification and all DNA based findings which are likely caused by a combination of factors such as primer efficiency and an incomplete reference database. Our findings underscore the critical importance of database completeness prior to implementing DNA-based techniques as standardized monitoring method, especially for metagenomics. Nevertheless, our findings emphasize that Oxford Nanopore metabarcoding proves to be a viable alternative to the conventional Illumina MiSeq metabarcoding platform for macrobenthos biodiversity monitoring.