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).