Protocol for designing and interpreting minigene assays to validate candidate splice altering variants

Abstract

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. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=197 SRC="FIGDIR/small/723105v1_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@1b6b44eorg.highwire.dtl.DTLVardef@1f795c4org.highwire.dtl.DTLVardef@189aa22org.highwire.dtl.DTLVardef@100a833_HPS_FORMAT_FIGEXP M_FIG C_FIG