Högskolan i Skövde

Freshwater ecosystems are increasingly threatened by anthropogenic stressors such as pesticide contamination, agricultural runoff, pollution, and climate change. Freshwater unionid mussels play a critical ecological role as filter feeders and bioindicators of aquatic ecosystem health; however, genomic resources for these organisms remain limited due to the large and repetitive nature of their genomes. This study aimed to sequence and assembled the genome of Anodonta anatina using Oxford Nanopore long-read sequencing technology while evaluating the effectiveness of two high-molecular-weight (HMW) DNA extraction methods. Fresh specimens of A. anatina were collected from Lake Vingsjön, Sweden, foot and gill tissues were processed using the Monarch HMW DNA Extraction Kit and the PacBio Nanobind PanDNA protocol. DNA quality and integrity were assessed using Qubit fluorometry, NanoDrop spectrophotometry, and pulsed-field gel electrophoresis (PFGE). Sequencing libraries were prepared using the Oxford Nanopore Ligation Sequencing Kit V14 and sequenced on MinION R10.4.1 flow cells. Sequencing quality was evaluated using NanoPlot, genome assembly was performed using Flye, and assembly quality was assessed using QUAST. PFGE and NanoPlot analyses demonstrated superior preservation of high molecular weight DNA and improved long-read distributions in PacBio-derived samples compared with Monarch-derived samples. The PacBio assembly generated a draft genome of approximately 1.38 Gb with an N50 value of 34,332 bp and a maximum contig length of 381,012 bp, whereas the Monarch assembly was substantially smaller and more fragmented. Although the assemblies remained incomplete relative to the estimated 3 Gb genome size, the study demonstrates the feasibility of nanopore-based genome assembly in A. anatina and highlights the importance of extraction methodology for long-read sequencing performance. These genomic resources provide a valuable foundation for future studies in freshwater mussel genomics, conservation genetics, and aquatic ecotoxicology.