RAD Paired-End Sequencing for Local De Novo Assembly and SNP Discovery in Non-model Organisms

R estriction-site A ssociated D NA (RAD) markers are rapidly becoming a standard for SNP discovery and genotyping studies even in organisms without a sequenced reference genome. It is difficult, however, to identify genes nearby RAD markers of interest or move from SNPs identified by RAD to a high-throughput genotyping assay. Paired-end sequencing of RAD fragments can alleviate these problems by generating a set of paired sequences that can be locally assembled into high-quality contigs up to 1 kb in length. These contigs can then be used for SNP identification, homology searching, or high-throughput assay primer design. In this chapter, we offer suggestions on how to design a RAD paired-end (RAD-PE) sequencing project and the protocol for creating paired-end RAD libraries suitable for Illumina sequencers.