DNA extraction and sequencing of historical specimens of Xerces Blue and Silvery Blue
All DNA extraction and initial library preparation steps (prior to amplification) were performed in the dedicated clean lab, physically-isolated from the laboratory used for post-PCR analyses. Strict protocols were followed to minimize the amount of human DNA in the ancient DNA laboratory, including the wearing a full body suit, sleeves, shoe covers, clean shoes, facemask, hair net and double gloving. All lab surfaces consumables, disposables, tools and instruments are wiped with bleach and ethanol, and UV irradiated before and after use.
DNA extraction was performed from 12 abdominal samples of Xerces Blue and Silvery Blue. One ml of digestion buffer (final concentrations: 3 mM CaCl2, % SDS, 40 mM DTT, 0.25 mg/ml proteinase K, 100 mM Tris buffer pH 8.0 and 100 mM NaCl) was added to each crushed butterfly residue, including an extraction blank, and incubated at 37 °C overnight (24h) on rotation (750-900 rpm). Next, DNA extraction was continued following the method proposed by Dabney et al. (2013)(25). Remaining butterfly sample was then pelleted by centrifugation in a bench-top centrifuge for 2 min at maximum speed (16,100 × g). The supernatant was added to 10 mL of binding buffer (final concentrations: 5 M guanidine hydrochloride, 40% (vol/vol) isopropanol, 0.05% Tween-20, and 90 mM sodium acetate (pH 5.2)) and purified on a High Pure Extender column (Roche). DNA extracts were eluted with 45 μL of low EDTA TE buffer (pH 8.0) and quantified using a Qubit instrument.
Following extraction, 100-200 ng of DNA extract was converted into Illumina sequencing libraries following the BEST protocol(26). Each library was amplified by PCR using two uniquely barcoded primers. After index PCR, libraries were purified with a 1.5x AMPure clean (Beckman Coulter) and eluted in 25 μl of low EDTA TE buffer (pH 8.0). Libraries were quantified using BioAnalyzer and sequenced by HiSeq 4000 (Illumina).