2.4 RAD-seq data
We extracted genomic DNA from either kidney or muscle tissue using Qiagen DNEasy Blood and Tissue kit (Qiagen, CA), following the manufacturer’s protocol. A total of 164 individuals had genomic DNA of sufficient quantity (> 50 ng) and quality needed for restriction site-associated sequencing (RAD-seq) as determined through gel electrophoresis (Davey et al., 2011). RAD-seq library preparation followed the BestRad protocol for Illumina sequencing as described in Ali et al. (2016). Briefly, genomic DNA (100 ng) was digested with 4.8 units of Sbf I-HF restriction enzyme (New England Biolabs NEB, R3632L) at 37°C for 1 h in a 12 µl reaction volume. Samples were heated to 65°C for 20 min and 4 µl of the indexed BestRad SbfI P1 RAD adapter (10 nM) was added to each sample. Ligation of inline barcoded P1 adaptors to digested genomic DNA was performed overnight at 20°C with 640 units of T4 DNA ligase (NEB, M0202M), followed by incubation at 20ºC for 60 minutes, then 65°C for 20 min. Following ligation, 10 µl of each sample in each 48 well plate was pooled into a single tube and cleaned using 1x Agencourt AMPure XP beads (A63881; Beckman Coulter). Pooled DNA for each plate was then resuspended in 100 µl low TE and sheared to an average fragment size of 500 base pairs using a Bioruptor NGS sonicator (Diagenode). Sheared DNA was then concentrated to 55.5 µl using Ampure XP beads and used as the template in the NEBNext Ultra DNA Library Prep Kit for Illumina (NEB E7370L; v.1.2). The standard NEBNext protocol for library preparation was followed except that we used custom P2 adaptors which were created by annealing a NEBNext Multiplex Oligo for Illumina (NEB, E7335L) to the oligonucleotide GATCGGAAGAGCACACGTCTGAACTCC AGTCACIIIIIIATCAGAACA*A (where * represents a phosphorothioate DNA base). In addition, instead of the USER® enzyme step, we used a universal P1 RAD primer (AATGATACGGCGACCACCGAGATCTAC ACTCTTTCCCTACACGAC*G) and a universal P2 RAD primer (CAAGCAGAAGACGGCATACG*A) during final amplification. The final RAD library was cleaned using AMPure XP beads and sequenced at the UC Berkeley QB3 Vincent J Coates Genome Sequencing Laboratory (GSL) on an Illumina HiSeq2500: Rapid Run Mode (Illumina, San Diego, CA, USA) using paired-end 100-bp sequence reads.