RADSEQ of Melanerpes carolinus
Genomic sampling and sequencing were performed using the NextRAD (Nextera-tagmented, Reductively Amplified DNA) method. It yields short sequences (~ 150 base pairs) flanked by engineered transposon cut sites, which results in thousands of loci distributed throughout the genome (Russello et al., 2015). This method requires small amounts of DNA (less than 50 ng; Russello et al., 2015), allowing the recovery of sequence data even from museum specimens with low DNA concentration. NextRAD genotyping-by-sequencing libraries were made from genomic DNA by SNPsaurus LLC (Oregon, USA) following Russello et al. (2015). To do this, genomic DNA (~10 ng) was fragmented with Nextera Flex reagent (Illumina, Inc), which also ligated short adapter sequences to the ends of the fragments. Fragmented DNA was amplified with one of the primers matching the adapter and extending 10 nucleotides into the genomic DNA with the selective sequence GTGTAGAGCC. The constructs were amplified by PCR using Phusion Hot Start Flex DNA Polymerase (New England Biolabs; Massachusetts, USA). PCR program consisted of an initial step of 3min at 72C, followed by 30s at 98C, and five denaturation cycles at 98C for 10s, annealing at 63C for 30s, and 3min extension at 72C. Amplicons were pooled and the resulting libraries were purified using AMPure XP beads (Agencourt Bioscience Corporation; Massachusetts, USA) at 0.7. Then, purified libraries were size-selected to 350-800 base pairs and sequenced on a HiSeq 4000 with one lane of 150 bp reads (Genomics Core Facility, University of Oregon).