Genotyping by Sequencing for Population Genetics

For each individual examined, we extracted total DNA from vouchered tissue samples using DNeasy tissue kits (Qiagen, Valencia, California, USA) following the manufacturer’s protocol. We sent DNA extracts to the Cornell Institute of Genomic Diversity (IGD) to collect data using Genotyping by Sequencing, a RAD-Seq method (Elshire et al., 2011 (link)). Briefly, the IGD digested DNA using PstI (CTGCAG) and ligated a sample-specific indexed adapter and common adapter to resulting fragments. The IGD pooled and cleaned ligated samples using a QIAquick PCR purification kit (Qiagen, Valencia, CA, USA), amplified the pool using an 18-cycle PCR, purified the PCR product using QIAquick columns, and quantified the amplified libraries using a PicoGreen assay (Molecular Probes, Carlsbad, California, USA). Based on the PicoGreen concentrations, the IGD then combined the samples for this project with unrelated samples and ran plates of 96 samples on a 100-base pair, single-end Illumina HiSeq 2000 lane (Illumina, San Diego, California, USA).