Targeted Long-Read RNA Sequencing Enhances Genetic Variant Interpretation

Overview

A novel targeted long-read RNA sequencing method, STRIPE, improves detection and interpretation of pathogenic variants in rare disease genes. This approach enhances molecular diagnosis rates by revealing complex RNA processing effects and linking variants to specific alleles.

Background

Current genetic testing methods such as exome and genome sequencing identify disease causes in only 20 to 50 percent of cases, leaving many patients without a molecular diagnosis. RNA sequencing offers additional insights by showing how genetic variants impact gene expression and splicing. However, standard short-read RNA sequencing has limitations in analyzing full-length transcripts and phasing variants. The STRIPE workflow was developed to address these challenges by focusing on disease-related genes and using long-read sequencing to analyze complete RNA transcripts.

Data Highlights

Key Findings

• STRIPE identified all previously reported pathogenic variants in patients with known genetic diagnoses.
• Long-read sequencing enabled phasing of variants across larger DNA regions, distinguishing whether variants are on the same or different alleles.
• More than half of splice site variants caused complex RNA changes beyond simple exon skipping, including early transcript termination via alternative polyadenylation.
• The method detected disease-causing variants in patients lacking prior genetic diagnoses, outperforming standard RNA sequencing approaches.
• Targeted sequencing achieved greater depth and improved detection of low-level transcripts in accessible tissues like fibroblasts.
• Limitations include restriction to genes included in the panel and the need for workflow standardization before clinical implementation.

Clinical Implications

STRIPE offers a promising tool to enhance molecular diagnosis in rare diseases by providing detailed insights into variant effects on RNA processing and phasing. Its targeted approach allows deeper sequencing of clinically relevant genes, potentially improving diagnostic yield in patients with inconclusive standard testing. Integration into clinical workflows will require further validation and standardization.

Conclusion

Targeted long-read RNA sequencing via STRIPE advances the interpretation of genetic variants by revealing complex transcript alterations and enabling allele-specific analysis, thereby supporting improved diagnosis of rare genetic disorders.

References

1. Science Advances 2024 -- Unlocking Hidden RNA Signals