DNA extraction and genotyping
We extracted DNA from blood samples collected using a Qiagen QIAmp DNA
Blood Mini kit (Qiagen, Valencia, CA, USA) following the manufacturer’s
protocols. Extracted DNA from samples were quantified using a NanoDrop
(Thermo Scientific 2000c) and a Qubit 2.0 fluorometer with the DNA HS
assay kit (Life Technologies). We also checked for DNA quality based on
migration on agarose gel, which allowed for the selection of samples
with appropriate DNA concentration (> 20 ng/µL) and
molecular weight (> 10000 bp). We sequenced 79
yellow-fronted and red-fronted tinkerbirds using a double-digest
restriction-site associated DNA lab protocol (Brelsford et al., 2016),
which incorporates elements of protocols from previous studies (Parchman
et al., 2012; Peterson et al., 2012). Briefly, genomic DNA was digested
with restriction enzymes SbfI and MseI and double-stranded adapters with
inline barcodes of 4 to 8 bp were ligated to the resulting fragments.
After magnetic bead purification, the fragments were amplified by PCR in
4 replicate reactions per individual sample and pooled. We implemented a
final purification and size selection of the pooled library using
magnetic beads in order to remove primer dimers [and fragments less
than 150 bp]. Our library was then and sequenced on two lanes on the
Illumina Hiseq X platform by Novogene Inc., with 150 bp paired-end
reads.