Population Genomics Analysis with RAD, Reprised: Stacks 2.

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.