Inverse Restriction Site-Associated DNA Sequencing (iRAD-seq).

Reduced representation sequencing (RRS), particularly through restriction site-associated DNA sequencing (RAD-seq), has been widely adopted for whole-genome genotyping due to its cost-effectiveness and cross-species applicability. Nevertheless, conventional RAD-seq approaches are constrained by intricate workflows and substantial labor intensity. These methods predominantly adhere to a "fragment selection precedes library construction" paradigm, wherein DNA fragments adjacent to restriction enzyme cleavage sites are specifically targeted. In contrast, we present an innovative strategy termed inverse restriction site-associated DNA sequencing (iRAD-seq), which implements a reversed workflow, "library construction precedes fragment selection," to enable efficient enrichment of DNA fragments not associated with restriction sites for genome-wide genotyping. This approach harnesses Tn5 transposase to concurrently fragment genomic DNA and ligate sequencing adapters, followed by pooled processing of hundreds of libraries under a unified batch restriction digestion step. The iRAD-seq workflow thereby achieves significant simplification and enhances operational efficiency in RAD-seq library preparation. Key features • iRAD-seq is a swift and simple RRS method based on Tn5 library construction and restriction enzyme digestion. • The library preparation for iRAD-seq adopts a strategy of library construction followed by fragment selection. • iRAD-seq enables effective reduction of genome complexity, with the extent of simplification flexibly tunable by adjusting the combination of restriction enzymes.