NovaSeq ddRAD of Euchloe ausonides insulanus
For restriction digests, template with a concentration greater than 15 ng/ (a majority of samples) was diluted in Tris buffer to 15 ng/, and any samples with less than 15 ng/ were used as-is. Each sample was digested individually in a reaction consisting of 0.9  CutSmart buffer, 0.28  Pst1 enzyme, 0.12  Mse1 enzyme, 1.7  PCR Grade water, and 6  of template DNA. Samples were digested in a 96 well plate on a thermal cycler at 37 for three hours, followed by storage at 4. A 1.5X ratio of Ampure beads:sample were added to each digest (13.5 ) for purification, and eluted in a volume of 22  Tris (of which 20  were collected) after two consecutive washes with 200  80% ethanol. Adapters for input into ligation reactions were constructed following Peterson et al. (2012). Size selection was performed with a ThermoFisher 2% size-select E-gel following the manufacturer instructions. Both ~200 bp and ~400 bp size fractions were targeted and ~45  of each collected. The ~200 bp fragment pool was chosen for pilot sequencing. Five replicate PCRs using four different dual indexed primer pairs (a total of 20 PCR reactions; Table S5) were performed using the following reaction conditions: 2  size elected template, 8  NEB OneTaq MMix, 0.8  pooled forward and reverse primers (10  stock concentration) and 5.2  PCR grade water. Thermal cycling consisted of 94 for 2min then 20 cycles of (94 for 30sec, 60 for 30sec, 68 for 45sec) 12 hold. This approach was intended to introduce sequence variation into the run. 10  of each PCR product was combined and purified using Ampure beads following previous steps. The size distribution of the library was assessed using a D-1000 Tapestation DNA screentape and diluted to 4 nM following Illumina recommendations. The final library was sequenced on a NovaSeq 6000.