Eopsaltria australis Targeted Locus (Loci)

Capture sequencing of wild samples of the Eastern Yellow Robin (Eopsaltria australis) collected through the entire species range. Targeted sequencing was performed via genomic hybrid capture approach with probes designed based on a draft of the Eastern Yellow Robin genome (Morales, Wang, Pavlova and Sunnucks, unpublished data).A total of 2000 anonymous target loci (240 bp) for hybrid enrichment were designed from the genome sequence at 4x coverage. Capture loci had 40% and 55% GC content, were low-copy, and the full set of pseudo-reads were mapped to the candidate sequences using a preliminary 25-kmer exact match and a final 72 of 120 consecutive bp match requirements. Assembly profiles were used to remove outlier loci by requiring five or six reads mapped and at least 96% average identity across mapped reads. These filters retained 1356 loci, of which 1000 were randomly selected to serve as targets for hybrid enrichment. Probes (120 bp each) were tiled uniformly at 2x density (3 probes per locus) to form the probe set.Following Lemmon et al. (2012), indexed libraries were prepared from genomic DNA on a Beckman-Coulter FXp liquid-handling robot, and enriched using an Agilent Sure Select enrichment kit, with 24 robin samples pooled per enrichment reaction. Following qPCR and Bioanalyzer-based quality control, libraries were sequenced on an Illumina 2500 lane with paired-end 150 bp reads and 8 bp indexing read.