A Practical and Cost-Effective Approach to Long-Fragment eDNA Sequencing for High-Resolution Genetic Diversity Assessment

Environmental DNA (eDNA) analysis is increasingly recognised as a valuable method for assessing genetic diversity. However, its resolution and applicability are limited by the short length of sequences that can be analysed (typically < 400 bp) and high analytical costs. This study developed a practical, low-cost long-fragment eDNA analysis method using commercial full-length plasmid sequencing via a nanopore platform and evaluated its effectiveness in assessing population genetic structure. 1 L of surface water was collected from 52 sites across Hokkaido, Japan, targeting Barbatula oreas. Two mitochondrial regions (ND5 and cyt b; approximately 1,000 bp each) were species-specifically amplified, circularised, and sequenced. Library preparation took 2.5 hours, with a total cost per sample of 4,390 JPY ({approx}25.55 EUR, {approx}29.87 USD). High-quality reads were obtained from 34 samples, allowing for the reconstruction of multiple haplotypes per region through haplotype phasing. The eDNA concentration required to achieve a 50% sequencing success was within a range easily attainable for common species. Phylogenetic analysis using 62 concatenated haplotypes (1,968 bp) obtained from each sample identified two clades and multiple regional subgroups, providing higher-resolution phylogeographic information than the previous study. Furthermore, the differentiation of each clade and group was suggested to reflect geological and climatic events. These results demonstrate the feasibility and utility of long-fragment eDNA analysis for evaluating genetic diversity, and its broad application is anticipated in ecological research, conservation management, and environmental policy formulation.