Restriction enzymes have been one of the primary tools in the population genetics toolkit for 50 years, being coupled with each new generation of technology to provide a more detailed view into the genetics of natural populations. Restriction site-Associated DNA protocols, which joined enzymes with short-read sequencing technology, have democratized the field of population genomics, providing a means to assay the underlying alleles in scores of populations. More than 10 years on, the technique has been widely applied across the tree of life and served as the basis for many different analysis techniques. Here, we provide a detailed protocol to conduct a RAD analysis from experimental design to de novo analysis—including parameter optimization—as well as reference-based analysis, all in Stacks version 2, which is designed to work with paired-end reads to assemble RAD loci up to 1000 nucleotides in length. The protocol focuses on major points of friction in the molecular approaches and downstream analysis, with special attention given to validating experimental analyses. Finally, the protocol provides several points of departure for further analysis.

RADseq dataset from two populations of the Patagonian blenny (Eleginops maclovinus). This fish species is native to the coastal regions of southern South America and is an outgroup to the cold-specialized radiation of Antarctic notothenioid fish (Chen et al. 2019). Sixty individuals from two populations in southern Chile (Valdivia n = 37, Puerto Natales n = 23) were processed into a single-digest RAD library (Baird et al. 2008; Etter et al. 2012) generated using the RE SbfI. The library was sequenced (2 × 150 bp) on a single lane of an Illumina NovaSeq6000 at the Roy J. Carver Biotechnology Center sequencing facility at the University of Illinois, Urbana-Champaign.

Files are a pair of gzipped FASTQ files containing raw Illumuna sequencing reads. Can be processed with standard bioinformatic software.