Development of a Comprehensive Analytical Workflow for eDNA Analysis of Vertebrates, Arthropods, and Mollusks

Environmental DNA (eDNA) analysis is a powerful tool for biodiversity monitoring, but conventional methods are often limited to specific taxonomic groups and short-read sequencing. This study aimed to enhance species detection using Oxford Nanopore long-read sequencing and 16Sar/br primers. Our results showed that MiFish primers effectively detected fish species, while COI primers primarily detected bacterial DNA, limiting eukaryotic identification. In contrast, 16Sar/br primers enabled the detection of diverse taxa, including fish, mollusks, and crustaceans.To improve accuracy, we expanded the mitochondrial 16S database, increasing its size from 4.7 GB to 7.0 GB and the number of sequences from 1.69M to 2.15M, leading to improved mollusk detection. Additionally, an automated analysis pipeline was developed, streamlining data processing. These findings highlight the potential of long-read sequencing and database enhancement for more comprehensive eDNA-based biodiversity assessments.