The heterogeneous stock (HS) is an outbred rat population derived from eight inbred rat strains. The population is maintained with the goal of minimizing inbreeding and maximizing the genetic diversity of the stock. To effectively utilize this rat strain for fine-scale genetic mapping, genotype data is necessary for large numbers of animals. A few genotyping microarrays have been created for rats; however, they were expensive and are no longer in production. Thus, to obtain high-density genome-wide marker data for genetic mapping, we have adapted genotype-by-sequencing (GBS) for use in rats. Here, we outline the laboratory and computational steps we took to design and optimize an efficient double digest genotype-by-sequencing (ddGBS) protocol for rats. We include a detailed protocol to perform ddGBS in rats. To analyze the ddGBS sequencing data, we evaluated multiple existing computational tools and designed a workflow that allowed us to call and impute over 3.7 million SNPs genome-wide in the HS. We also compared various rat genetic maps for use in imputation, including a recently developed map specific to the HS. Using the pipeline, we obtained concordance rates of 99% with data from a rat genotyping array. The computational pipeline that we have developed can be easily adapted for use in other species.### Competing Interest StatementThe authors have declared no competing interest.

Adapting genotyping-by-sequencing and variant calling for heterogeneous stock rats