Targeted long-read RNA sequencing for rare disease diagnosis and variant interpretation.

Diagnosing rare genetic diseases remains a major challenge despite widespread clinical testing. Long-read RNA sequencing (RNA-seq) offers a powerful approach to capturing the effects of genetic variants on the transcriptome, yet challenges with sequencing coverage, cost, tissue selection, and scalability have limited its clinical adoption. To address this, we developed STRIPE (Sequencing Targeted RNAs Identifies Pathogenic Events), a targeted long-read RNA-seq-based strategy for rare disease diagnosis and variant interpretation. STRIPE enables deep sequencing of full-length transcripts for any customized disease-specific gene panel such that a wide range of clinically informative readouts, including transcript aberrations and sequence variants, can be detected at haplotype-level resolution. Applying STRIPE to 88 individuals spanning two major rare disease groups, we accurately reidentified known pathogenic variants and revealed their transcript consequences, including many unexpected ones. For 8 of 15 splice site region variants, we observed more complex RNA processing defects beyond single exon skipping or cryptic splice site activation. Notably, we find that donor splice site variants frequently activate cryptic intronic polyadenylation sites, leading to premature transcript termination. Leveraging unique strengths of long-read RNA-seq, STRIPE also resolved variants of uncertain significance and uncovered disease-causing variants in five previously undiagnosed individuals. Overall, STRIPE is a powerful, adaptable, and scalable strategy with broad potential to improve clinical variant interpretation and advance genetic diagnosis of rare diseases.