ddRadSeq of Gyrinophilus porphyriticus
DNA was extracted from tissue samples using Nexttec DNA Isolation Systems Tissue and Cells kits (Hilgertshausen, Germany). Genomic DNA quality was visualized on an agarose gel and each sample was quantified using a BioTek Synergy HT Microplate reader. Libraries were prepared following the 2RAD/3RAD protocol developed by the EHS DNA Lab at the University of Georgia (Bayona-Vsquez et al., 2019). Briefly, we used the restriction enzymes BspDI and SbfI to digest 100 ng of genomic DNA per individual, immediately followed by adapter ligation. The use of 8 unique barcodes in the P1 adapter and 12 unique barcodes in the P2 adapter allowed us to pool 96 individuals per PCR. We used indexed iTru PCR primers for amplification, including a random 8 bp sequence in the P2 primer to enable detection of PCR duplicates (Schweyen et al., 2014). We then performed sequence capture using baits manufactured by Arbor Biosciences (MyBaits, Ann Arbor, MI, USA) to target 520 variable RAD loci identified in a previous experiment (Addis & Lowe, 2022).