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.