Evaluating eDNA metabarcoding methods for marine vertebrate monitoring

Résumé fourni par la source

Environmental DNA (eDNA) is rapidly becoming a valuable tool for conducting biodiversity research, including studies on marine vertebrates, and metabarcoding of eDNA enables the characterization of biological communities. However, methodological variation across workflow stages can influence results, highlighting the need for standardized and accessible protocols. In this study, two eDNA extraction strategies were evaluated, the performance of two DNA polymerases differing in proofreading capacity was tested, and the Marine Vertebrate eDNA Metabarcoding bioinformatics pipeline (MVeM)—an open-source, adaptable, and reproducible workflow for sequence processing and taxonomic assignment–was developed. For extraction, the standard DNeasy PowerWater Sterivex protocol was applied, and a preliminary bead-beating homogenization step performed on filter material prior to the PowerWater protocol was additionally tested, as was the recovery of free extracellular DNA in controlled seawater samples. Direct extraction from intact filters yielded higher DNA concentrations and amplicon sequence variant (ASV) richness than the bead-beating homogenization method, whereas both approaches effectively captured marine vertebrate diversity. The recovery of free extracellular DNA remained low with both extraction methods. During library preparation and sequencing, the non-proofreading polymerase generated more cetacean ASV reads in mock communities, suggesting that it is a cost-effective option for monitoring this group, whereas the proofreading enzyme improved the amplification of fish taxa. The MVeM pipeline integrates stringent sequence filtering, the Lowest Common Ancestor approach for ambiguous matches, a geographic exclusion list, and contamination control, enabling accurate, biologically realistic, and reproducible taxonomic assignments for marine vertebrate eDNA. Overall, combining appropriate extraction strategies, polymerase selection, and a robust bioinformatics framework provides a reliable, adaptable, and accessible approach for marine vertebrate biodiversity monitoring. Graphical abstract :

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