Whitford, W.; Musgrave, S. M.; Snell, R. G.; Jacobsen, J. C.

Variants affecting RNA splicing are a major contributor to human disease, yet the consequences of variants outside of the canonical splice motifs are often difficult to determine. Here, we present a protocol for minigene-based evaluation of candidate splice-altering variants. The methodology described includes locus-specific insert design, commercial gene fragment synthesis, and long-read sequencing. The combined approach enables rapid assay development and nucleotide level resolution of the effect on splice isoforms in vitro, providing a scalable framework for functional validation of predicted cryptic splice variants.