The study of genomic parallelism in three-spined stickleback using RAD sequencing

We performed a genome-wide scan of independently derived freshwater stickleback populations from Alaska, British Columbia, Iceland and Scotland (island of North Uist) using restriction site associated DNA (RAD) sequencing on 1 397 individuals from 73 independent lakes. Genomic DNA was purified from 10 to 21 individuals from each of the populations, chosen to represent a widely distributed subset of the most environmentally and phenotypically variable lakes. Extracted genomic DNA was normalized to a concentration of 25 ng /µL in 96-well plates.In 2014 we conducted RAD sequencing on samples from North Uist and from Iceland. Sequencing libraries were prepared following the modified and processed into RAD libraries according to Etter et al. (2011), using the restriction enzyme SbfI-HF (NEB). Each sample was individually ligated to adaptors with 6bp in-line barcodes and multiplexed in libraries of 192. We sequenced these RAD libraries in four lanes on an Illumina HiSeq sequencer at the University of Oregon, producing 100-bp single-end reads. In 2015 we conducted RAD sequencing on samples from British Columbia and from Alaska. Sequencing libraries were prepared following the modified single-digest RAD protocol of Ali et al. (2016), using the restriction enzyme SbfI, which digests DNA at an 8bp recognition sequence and is expected to cut roughly 22,000 locations across the stickleback genome. Each sample was individually labeled using one of 96 unique 8bp barcodes, and each pool of 96 samples received a different 8bp inline index using the NEBNext Ultra DNA Library Prep Kit for Illumina (Ali et al. 2016). We sequenced these RAD libraries in two lanes of Illumina NextSeq high-output sequencing at the University of Oregon, using paired-end 75bp reads. After filtering for the presence of a correct index, barcode, and SbfI cut site, these two lanes of sequencing produced a total of 256 million and 267 million read pairs, respectively.