Long-Read RNA Sequencing Platform Helps Diagnose Rare Diseases

For the first time, Children’s Hospital of Philadelphia scientists have applied long-read RNA sequencing at scale to reveal disease-causing variants in pediatric patients with a range of rare disorders.

The platform is called STRIPE (Sequencing Targeted RNAs Identifies Pathogenic Events). By developing a versatile, low-cost technology and a novel diagnostic workflow, the researchers identified diagnoses missed by other genetic tests, while also uncovering unexpected molecular insights into previously identified pathogenic variants.

The findings, published in Science Advances, represent work almost 25 years in the making, enabled by CHOP’s collaborative research environment and unparalleled expertise in rare diseases.

“This study highlights the transformative potential of long-read RNA sequencing in advancing the diagnosis of rare diseases,” said the study’s lead senior author Yi Xing, PhD, Associate Chief Scientific Officer for Omics, Technology & Engineering and Francis West Lewis Chair in Computational and Genomic Medicine. “Going forward, this platform could be applied to diagnose all sorts of rare genetic disorders, which could then be linked to precision therapies.”

Long-Read RNA Sequencing: Opportunities and Challenges

Around 300 million people worldwide live with rare diseases, which often cause severe health problems, early death, and heavy financial burdens. Despite widespread clinical testing, less than 30% of patients with rare diseases receive a molecular diagnosis, according to the New England Journal of Medicine, underscoring a need for more advanced diagnostic tools.

“High-throughput DNA sequencing is the primary technology for diagnosing rare genetic disorders,” Dr. Xing said. “However, even after DNA sequencing, the majority of patients remain undiagnosed.”

A key factor underlying this diagnostic gap is that many genetic variants cause disease by disrupting how RNA molecules are transcribed and processed, so their effects cannot always be understood from DNA sequencing alone.

As a result, researchers and clinicians are increasingly turning to RNA sequencing to better interpret how genetic variants alter gene activity and function to cause disease. While traditional RNA sequencing methods have drawbacks, long-read RNA sequencing has emerged as a powerful technology for RNA analysis. By directly sequencing full-length RNA molecules end-to-end, the method offers the potential to transform RNA-guided interpretation of genetic variants.

However, the clinical adoption of long-read RNA sequencing has been limited by lower throughput and high costs. Also, because RNA-based diagnostics often rely on clinically accessible tissues where disease-relevant genes may be expressed at low levels, deeper sequencing is frequently required for informative results.

“A major challenge in RNA-guided rare disease diagnostics is that disease-relevant tissues are often difficult to obtain from patients,” said co-senior author Rebecca Ganetzky, MD, an Attending Physician and clinical geneticist at CHOP.

These factors impede scaling long-read RNA sequencing in clinical settings and constrain its diagnostic potential, Dr. Ganetzky said.

Giving Answers to the Undiagnosed

To overcome these challenges, Dr. Xing and his collaborators developed STRIPE — a targeted long-read RNA sequencing-based platform that enables deep sequencing of full-length RNA molecules for any customized disease-specific gene panel. The CHOP team aptly named the STRIPE technology, giving a nod to the zebra — a widely recognized symbol of rare disease.

A key advantage of the STRIPE platform is that it enables high-quality analysis of RNA from clinically accessible tissues, such as skin fibroblasts and blood, while still capturing the disease-relevant signals needed to interpret the RNA-level effects of genetic variants, Dr. Xing said.

Their work builds upon CHOP researchers’ prior development of TEQUILA-seq, a scalable and low-cost technology for sequencing full-length RNA molecules.

“TEQUILA-seq was designed to make targeted long-read RNA sequencing cost-effective and scalable,” said co-senior author Lan Lin, PhD, Assistant Professor of Pathology and Laboratory Medicine, who developed the TEQUILA-seq technology. “With a much lower RNA-to-data cost, STRIPE enables ultra-deep full-length RNA sequencing of disease-relevant genes at a scale that is practical for clinical applications.”

To evaluate the platform, researchers used STRIPE to test RNA samples from 88 children across two groups of rare diseases: congenital disorders of glycosylation (CDG) and primary mitochondrial diseases (PMD). These disease classes include many genetically diverse conditions, with new disease-causing variants continuing to be discovered, making them well-suited to assess the effectiveness of STRIPE, said co-senior author Andrew C. Edmondson, MD, PhD, Founding Director of the CDG Clinic and an Attending Physician in the Division of Human Genetics.

Applying STRIPE to patients, the researchers revealed several key technological and conceptual advances that push the boundaries of current RNA-based diagnostic technologies.

“CDG patients with high unmet diagnostic needs were given access to a novel technology, ending their diagnostic odyssey and facilitating them to appropriate clinical care,” Dr. Edmondson said.

The research team confirmed that STRIPE can read RNA molecules from parts of the genome that are highly duplicated or repetitive. While many routine tests miss changes in these duplicated regions, STRIPE allowed CHOP researchers to make a genetic diagnosis for a patient who had been undiagnosed for more than 10 years.

STRIPE is also able to read whole RNA molecules end-to-end, so the researchers could see exactly how a DNA change affects the full RNA product. This allowed the team to reclassify some DNA changes of uncertain clinical significance as disease-causing and find harmful regulatory changes buried inside exons or introns. As a result, the team was able to give clear molecular diagnoses to four more patients who previously had unexplained conditions.

The researchers’ tests revealed that, even for some pathogenic variants already identified in previously diagnosed patients, their data showed more complicated problems than expected. For example, the cell sometimes keeps introns that should be removed, skips multiple exons, or terminates RNA molecules prematurely. These complicated changes give new clues about how these variants cause disease.

“This knowledge can empower families to understand the cause of a child’s complex disease, to test which other family members may be disease carriers, and to pursue a future family planning option of preimplantation genetic testing,” said co-senior author Marni Falk, MD, an Attending Physician and Executive Director of the Mitochondrial Medicine Frontier Program at CHOP.

Since its development, STRIPE has been deployed in more than 500 patients across multiple clinical programs at CHOP, demonstrating its real-world potential and scalability for rare disease diagnostics.

A Bridge from Diagnosis to Precision Therapies

STRIPE reflects a scientific thread that spans more than two decades. The idea began with a diagram Dr. Xing drew as a graduate student in 2001.

“This deceptively simple diagram, now known as a splicing graph, captured a complex idea: Genes can produce many different RNA isoforms through alternative splicing, and this combinatorial complexity can be represented and modelled as a graph,” Dr. Xing said.

Over the next two decades, the field of transcriptomics transformed. First came gene expression microarrays, then short-read RNA sequencing, and now long-read RNA sequencing. Each wave brought the field closer to something that was once aspirational: a direct, high-resolution view of full-length RNA molecules.

“It’s incredibly meaningful to see this arc come full circle, especially knowing these early computational ideas from 25 years ago can now help diagnose children who previously had no answers,” Dr. Xing said.

Dr. Xing credited CHOP’s rare disease ecosystem, along with its vision for research-driven precision medicine, as critical to this work.

“STRIPE provides a bridge from genetic diagnosis to disease mechanism to targeted therapies,” Dr. Xing said. “I feel very fortunate and profoundly grateful to be at CHOP, where the integration of research and clinical care, together with major institutional investments like the Omics Initiative, has enabled us to translate these ideas into real impact for patients and families.”

Reference: Wang R, Wang F, DeBruyne N, et al. Targeted long-read RNA sequencing for rare disease diagnosis and variant interpretation. Sci Adv. 2026;12(16):eady9895. doi: 10.1126/sciadv.ady9895

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