title: ddRAD-seq of western fence lizard, Sceloporus occidentalis
design: We followed Peterson et al.'s (2012) method for genomic library preparation, with some modifications. For each individual we extracted high-molecular weight genomic DNA using a standard phenol-chloroform extraction protocol (Tsai et al. 2019). We measured DNA concentrations using a Qubit fluorometer, and for each sample we digested 0.5 g of DNA for 3 hours with SbfI and NIaIII restriction enzymes. We then purified these fragments with Agencourt AMPure beads before ligation of barcoded Illumina adaptors onto the fragment ends. All barcodes differed by at least two base pairs to reduce duplexing error rates. We then pooled equimolar amounts of each sample before conducting size selection using a BluePippin Prep to select fragments between 400 and 550 bp in length. We used proofreading Taq and Illuminas index primers for final library amplification for 8-10 cycles to reduce PCR bias. We quantified the final library concentration using a Qubit fluorometer at high sensitivity. Samples were packed on dry ice and sent to the Vincent J. Coates Genomics Sequencing Lab at UC Berkeley for sequencing. Quantitative PCR was used to determine concentration of adapter-associated fragments, and a BioAnalyzer run was used to confirm fragment sizes as a quality control measure prior to sequencing. Final libraries were sequenced (100-bp or 150-bp, single-end runs) in an Illumina HiSeq 4000 or NovaSeq SP lane for a total of four sequencing lanes.