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

DNA quality and quantity metrics (Microsoft Excel worksheet with 3 sheets, one per species). 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 (5 or 7 years for Southern Brook Lamprey). Four metrics were collected: DNA quantity (measured using Qubit 3.0 instrument with dsDNA high sensitivity kit); DNA purity measured by A260/A280 ratio (measured using NanoDrop 2000 spectrophotometer); DNA purity measured by A260/A230 ratio (measured using NanoDrop 2000 spectrophotometer); and percent fragments >= 300 base pairs (measured using ImageJ). For spectrophotometric absorbance ratios, an A260/A280 ratio of 1.8 and an A260/ A230 ratio of 2.0 were considered to represent “pure” DNA (i.e., free of contaminants). For analyses of DNA purity, deviations from 1.8 for the A260/A280 ratio and from 2.0 for the A260/A230 ratio were used as the response variables, such that larger deviation values corresponded to less-pure DNA. Please see attached README.md file for additional information