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

Abstract

Recent technological developments may facilitate the description of evolutionary relationships and population genetic structure, and other information relevant to fisheries management 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 sufficient‐quality DNA is recovered. We compared two protocols developed to extract DNA from formalin‐fixed tissues using specimens of three freshwater fishes: 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 (five or seven years for Southern Brook Lamprey). Likelihood models generally did not detect DNA quantity differences between extraction protocols or tissue types; however, 6.0‐8.7x more DNA was recovered from Slimy Sculpin caudal fins than 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. Our results suggest that the best tissue for sampling may depend on the quality metric most important for a study's objectives, and that omitting time consuming tissue wash steps can yield DNA of quantity and quality comparable to more complex methods. The DNA extracted from most samples, regardless of species, using both protocols met quantity and quality thresholds 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.

Bringing partners together: A symposium on native lampreys and the Pacific Lamprey Conservation Initiative

“…The unique body form and life-history diversity of lampreys commonly require modifications of standard techniques developed for bony fishes (e.g., Moser et al 2007;Dawson et al 2015;Lampman et al 2021), and the development and testing of new methods has contributed to the increase in lamprey publications since the early 2000s (Figure 1). Papers in this issue report on critical conservation needs, including methods evaluating phylogenetic standing (Brino et al 2023, this special section) and estimating lamprey size and age (Pelekai et al 2023, this special section). Litts et al (2023, this special section) and Spanos et al (2023, this special section) describe and evaluate collection and tagging approaches for field studies of adult lampreys.…”

“…Papers in this issue report on critical conservation needs, including methods for evaluating phylogenetic standing (Brino et al. 2023, this special section) and estimating lamprey size and age (Pelekai et al. 2023, this special section).…”

Buffered Lugol's Iodine Preserves DNA Fragment Lengths

“…By adopting protocols that mitigate formalin-induced alteration, such as the use of heat and alkali treatments to break protein–DNA cross-linkages caused by formalin exposure ( Hykin et al. 2015 ; Brino et al. 2023 ), researchers now routinely recover genomic information from specimens that were once considered too degraded or altered for genetic analysis ( Zimmermann et al.…”