GBS of Passerina cyanea x amoena: adult male pectoralis
We extracted genomic DNA from pectoralis tissue using a Qiagen DNeasy blood and tissue kit and a QIAcube robot following the manufacturer's standard extraction protocol (Qiagen, Inc.). One library was prepared using a genotyping-by-sequencing protocol. DNA samples were fragmented and prepared with the restriction endonucleases EcoRI and MseI. Fragments shorter than 275 base pairs and longer than 375 base pairs were excluded. Ten base pair barcodes denoting an individual bird were ligated to each fragment. Prior to sequencing, a 10\% PhiX spike was added to ensure sufficient variability in the first 25 bases of neighboring clusters. Prepared fragments were then multiplexed and amplified by PCR. The library was sequenced on a NextSeq 2000 P3 cartridge to produce 1 billion 138 base pair single-end reads. All sample quality control, library preparation, and sequencing was performed at the University of Wyoming Genome Technologies Laboratory in Laramie, Wyoming.