Long-Read RNA Sequencing Platform Improves Rare Disease Diagnosis

Standard DNA-based tests can miss variants that disrupt RNA transcription and processing, limiting diagnostic yields to only 20% to 50%. Clinicians therefore seek tools that directly interrogate full-length RNA to resolve uncertain findings from prior testing. A new study shows a targeted long-read RNA sequencing platform can uncover disease-causing variants and deliver molecular diagnoses that standard approaches missed.

At Children’s Hospital of Philadelphia (CHOP), researchers developed STRIPE (Sequencing Targeted RNAs Identifies Pathogenic Events), a targeted long-read RNA sequencing platform for disease-specific gene panels. Built on CHOP’s prior TEQUILA-seq technology, STRIPE was designed to address accuracy, cost, and scalability barriers that have limited wider use of long-read RNA sequencing in rare disease evaluation. The platform enables deep sequencing of full-length RNA molecules for any customized disease-relevant panel.

Unlike traditional methods that fragment RNA before sequencing, STRIPE captures transcripts end-to-end to connect disease-associated variants with abnormal RNA processing events on the same molecule. The approach supports high-quality analysis from clinically accessible tissues, including skin fibroblasts and blood, while preserving disease-relevant signals needed to interpret variant effects. Together, the targeted design and long-read capability allow ultra-deep coverage of selected genes at practical scale.

The team evaluated STRIPE across two rare disease groups extensively studied at CHOP: congenital disorders of glycosylation (CDG) and primary mitochondrial diseases (PMD). In total, 88 individuals spanning these disorders and healthy controls were analyzed. The platform accurately re-identified known pathogenic variants and clarified the RNA-level consequences of variants of uncertain significance.

Importantly, STRIPE uncovered new disease-causing variants in five previously undiagnosed patients, enabling clinicians to establish molecular diagnoses after standard testing had failed. Since its development, the platform has been applied to more than 500 patients across multiple CHOP clinical programs, indicating real-world scalability for rare disease diagnostics. The study, “Targeted long-read RNA sequencing for rare disease diagnosis and variant interpretation,” was published in Science Advances on April 15.

“TEQUILA-seq was designed to make targeted long-read RNA sequencing cost-effective and scalable. With an RNA-to-data cost of around $100 per sample, STRIPE enables ultra-deep, full-length RNA sequencing of disease-relevant genes at a scale that is practical for clinical applications,” said Lan Lin, Ph.D., assistant professor of Pathology and Laboratory Medicine at CHOP and developer of the TEQUILA-seq technology.

“By directly revealing how genetic variants disrupt RNA molecules, STRIPE provides a bridge from genetic diagnosis to disease mechanism to targeted therapies. More broadly, this work reflects a long-standing effort to interpret genetic variants at the level of full-length RNA molecules, and we believe STRIPE can serve as a foundation for RNA-based precision medicine in rare diseases, linking precision diagnostics to precision therapeutics,” said Yi Xing, Ph.D., Associate Chief Scientific Officer for Omics, Technology & Engineering and Francis West Lewis Chair in Computational and Genomic Medicine at CHOP.

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