Protocol for Designing and Interpreting Minigene Assays to Validate Candidate Splice-Altering Variants Using Long-read Sequencing.

Variants affecting RNA splicing are a major contributor to human disease, yet determining the functional consequences of candidate splice-altering variants remains challenging, particularly for variants located outside canonical splice motifs. Although computational prediction tools have improved identification of potentially spliceogenic variants, experimental validation is often required to determine their impact on transcript processing. Minigene assays provide a practical and controlled approach for functional assessment, particularly when patient-derived RNA is unavailable or genes are not expressed in accessible tissues. This article describes a workflow for the design, generation, and interpretation of minigene assays for evaluation of candidate splice-altering variants. The protocol includes locus-specific insert design, commercial gene synthesis, transient transfection into cultured cells, transcript amplification, and long-read Oxford nanopore sequencing for full-length splice isoform analysis. Particular emphasis is placed on preserving sufficient local splicing context while maintaining constructs compatible with efficient plasmid synthesis and mammalian cell transfection. Compared with traditional minigene workflows relying primarily on gel electrophoresis and Sanger sequencing, long-read sequencing enables direct single-molecule resolution of complete transcript isoforms, including combinations of exon skipping, intron retention, cryptic splice site activation, and partial exon usage. This workflow provides a scalable and practical framework for functional validation of candidate splice-altering variants in research and diagnostic settings. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Design and generation of minigene constructs Basic Protocol 2: Cell transfection, RNA extraction, and cDNA amplification Basic Protocol 3: Long-read sequencing and analysis of splice isoforms.