Double Digest Restriction-Site Associated DNA Sequencing (ddRAD-Seq).

ddRAD-Seq is a reduced representation sequencing technique that results in sequence datasets that can be compared and used to identify SNPs. We present an improved ddRAD-Seq protocol that increases efficiency and decreases the time to complete a ddRAD-Seq experiment. It utilizes selected restriction enzyme digestion fragments, quick acting ligases that are neutral with the restriction enzyme buffer eliminating buffer exchange steps, and adapters designed to be compatible with Illumina index primers. Enzyme deactivation steps are eliminated, library amplification and barcoding are completed in one PCR step, highly-efficient and precise size selection with BluePippin system and cleanup steps using magnetic beads are consolidated at the end of the library generation step. The SNPs that we identified using this streamlined protocol were validated in population and evolutionary studies of cotton (plant) (Magbanua et al., Anal Biochem 662:115001, https://doi.org/10.1016/j.ab.2022.115001 , 2023) and rohu carp (animal) (Arick et al., G3 (Bethesda) 13, https://doi.org/10.1093/g3journal/jkad009 , 2023).