Comparing DNA extraction methods for successful PacBio HiFi sequencing: a case study of the freshwater mussel Anodonta anatina (Bivalvia: Unionidae)

High-quality reference genomes are increasingly recognized as essential resources in biodiversity genomics and conservation. However, successful DNA extraction and long-read sequencing remain highly organism-dependent. Molluscs, a diverse phylum of invertebrates, pose particular challenges due to inhibitory compounds and degraded or fragmented DNA, often necessitating careful optimization of extraction protocols. Here, we present a case study on the freshwater mussel Anodonta anatina (Bivalvia: Unionidae), evaluating two preservation methods, six DNA extraction protocols, and two post-extraction clean-up steps for their effects on DNA quality and PacBio HiFi sequencing yield from a single individual. The PacBio Nanobind and CTAB protocols produced high-quality DNA from fresh foot tissue but performed poorly on flash-frozen tissue, which is the most common preservation method. Post-extraction clean-up generally degraded DNA and did not improve sequencing yield. Unexpectedly, the column-based Omega Mollusc Kit—although not designed for high-molecular-weight DNA—outperformed the PacBio-recommended Nanobind kit and the manual CTAB method on flash-frozen tissue. It generated high DNA quantity and purity, sufficient integrity for HiFi sequencing, and effective contaminant removal. While the resulting DNA may be too fragmented for Oxford Nanopore sequencing, the Omega Mollusc Kit offers a practical, cost-effective first approach for testing DNA extraction and PacBio sequencing in new mollusc genome projects of relatively large species when tissue is flash-frozen. When fresh tissue is available, Nanobind or CTAB remain the recommended options. This strategy could reduce the need for extensive protocol optimization and facilitate future mollusc genomics efforts.