Unlocking the genomes of formalin-fixed freshwater fish specimens: An assessment of factors influencing DNA extraction quantity and quality

Objective: Recent technological developments may facilitate the description of evolutionary relationships and population genetic structure as well as other information relevant to fisheries management by using readily available natural history collections. Contemporary sequence capture and short-read sequencing methods offer opportunities to analyze highly fragmented DNA from formalin-fixed specimens so long as enough DNA of sufficient quality is recovered.Methods: We compared two protocols developed to extract DNA from formalin-fixed tissues using specimens of three freshwater fishes: the Southern Brook Lamprey Ichthyomyzon gagei, Slimy Sculpin Cottus cognatus, and Brown Trout Salmo trutta. Extractions were attempted using hot alkali digestion with and without buffer wash pretreatments to compare the DNA concentration, purity, and fragment length of DNA recovered between extraction protocols, tissue types (muscle and caudal fin tissue for Brown Trout and Slimy Sculpin), and preservation periods (5 or 7 years for Southern Brook Lamprey).Result: Likelihood models generally did not detect DNA quantity differences between extraction protocols nor tissue types; however, 6.0-8.7x more DNA was recovered from Slimy Sculpin caudal fins than from muscle tissue. Extraction protocol had mixed effects on DNA purity; the wash protocol outperformed the no-wash protocol for Slimy Sculpin and Brown Trout, but the reverse was true for the lamprey. Purer DNA was recovered from the caudal fins; however, fragment lengths were generally greater from muscle tissue for both ray-finned species.Conclusion: Our results suggest that the best tissue for sampling may depend on the quality metric considered most important for a study's objectives and that omitting time-consuming tissue wash steps can yield DNA of quantity and quality comparable to DNA from more complex methods. Regardless of species, the DNA extracted from most samples using both protocols met quantity and quality thresholds that are likely to result in short-read sequencing success. These results provide optimism for unlocking the wealth of genetic information in natural history collections for use in fisheries management and conservation genomics.