RADseq processing, loci discovery and variant calling
DNA was extracted from blood stored in SET buffer with salt extraction
protocol (Aljanabi & Martinez, 1997). Quality controls were performed
with QUBIT to ensure sufficient starting material.
Restriction-associated DNA tags were obtained with double-digesting the
DNA of 388 owls with PstI/BamHI restriction enzymes following a
customized version of Petersen et al (2012). Library construction,
sequencing, demultiplexing were performed by Bioname©, Finland. Quality
checks were performed with FASTQC (Andrews, 2014). We utilized the new
assembly as a reference for mapping the RADseq reads. Mapping was
performed with bwa-mem2 with a minimum mapping quality of 20
incorporating both single and paired-end reads and discarding unmapped
reads (Li, 2013). Coordinates-alignment files were fed into Stacks v2.4
pipeline for variant calling (Rochette, Rivera‐Colón, & Catchen, 2019).
We constructed 6 different SNP panels in order to explore the impact of
filtering and subsequent captured loci in exploring associations with
colour phenotypes. Specifically, we tested filters on minor allelic
frequency (MAF = 0.01; MAF = 0.05), loci presence (R) within the
population (no filter; R = 0.5; R =0.8), while holding a constant
maximum heterozygosity of 0.50 and always collecting one random SNP per
RADtag. Due to the expected association between colour and genes
involved in the melanin-production pathway mentioned above, we tracked
the number of reads mapped to those regions across the different
filtering steps. We accounted for RADseq reads mapped within contigs
where those genes where found, within the respective coding sequence, as
well as in a range of +/- 20Kb flanking the coding sequence. We further
calculated the percentage (in base pairs) of coding sequences covered by
our double-digest RAD-sequencing strategy.