RAPID: a targeted long-read RNA workflow for functional resolution of splicing variants in rare disease

Abstract Background Molecular diagnosis of rare disease plateaus at ~ 50%, partly due to technical limitations of short-read sequencing and the persistent challenge of interpreting variants of uncertain significance. Splice-altering variation represents a major source of unresolved cases, yet functional assessment remains difficult in routine practice. Methods We developed a fully modular, sample-to-answer workflow for targeted long-read RNA sequencing using Oxford Nanopore Technologies and applied it to six unsolved cases with suspected monogenic neurometabolic disease. Candidates were selected after WES/WGS and multidisciplinary team review indicating ≤ 5 genes of interest. The workflow was designed to be diagnostically deployable, enabling near-full-length transcript assessment from accessible tissues without reliance on large control cohorts. Results Targeted long-read RNA sequencing yielded actionable findings for all six probands. We confirmed pathogenic splice disruption in two cases, prompted gene exclusion in one case, and generated RNA-level evidence prioritising further DNA investigation in three cases. Across these scenarios, long-read RNA sequencing provided direct, mechanism-level insight that either resolved diagnosis or refined variant interpretation. The workflow provided near-full-length isoform structures with reproducible single-sample interpretation and produced informative results within a clinically relevant timeframe using standard long-read sequencing infrastructure. Conclusions Targeted long-read RNA sequencing offers rapid, cost-effective functional evidence to resolve variants of uncertain significance, direct DNA follow-up, and support timely diagnosis in rare disease. The RAPID workflow demonstrates that long-read RNA sequencing can be implemented within existing diagnostic infrastructure and provides a scalable route to routine transcript-level assessment in clinical genomics.