Abstract
Identification of sequence variants that create or eliminate splice sites has proven to be a significant challenge and represents one of many roadblocks in the clinical interpretation of rare genetic variation. Current methods of identifying splice altering sequence variants exist, however, these are limited by an imperfect understanding of splice signals and cumbersome functional assays. We have recently developed a computational tool that prioritizes putative splice-altering sequence variants, and a moderate-throughput minigene assay that confirms the variants which alter splicing. This bioinformatic strategy represents a substantial increase in accuracy and efficiency of historical in vitro splicing assays. In this unit we give detailed instructions on how to organize, run, and interpret various features of this protocol. We expect that splice-altering variants revealed through this protocol can be reliably carried forward for further clinical and biological analyses.