Clinical validation of RNA sequencing for Mendelian disorder diagnostics

Despite rapid advancements in clinical sequencing, over half of diagnostic evaluations still lack definitive results. RNA-seq has shown promise in research settings for bridging this gap by providing essential functional data for accurate interpretation of diagnostic sequencing results. However, despite advanced research pipelines, clinical translation of diagnostic RNA-seq has not yet been realized. We have developed and performed validation for a clinical diagnostic RNA-seq test in a CLIA laboratory for individuals with suspected genetic disorders who have existing or concurrent comprehensive DNA diagnostic testing. Accepted sample types include RNA from fibroblast or blood samples. The clinical interpretation of RNA-seq results revolves around detecting outliers in gene expressions and splicing patterns. The clinical validation involves 150 samples, including samples accessible to most investigators with a benchmark potential, samples from apparently healthy individuals, and samples with a molecular genetic diagnosis characterized by abnormal RNA findings. We performed optimization and familiarization and set up passing criteria for quality control. We established provisional expression and splicing benchmarks using short-read and long-read RNA-seq data of the HG002 lymphoblastoid sample from the Genome in a Bottle Consortium. Our validation data achieved sensitivity and specificity higher than 99% against the benchmarks. For clinical validation, we first established the reportable range for each gene and each junction based on our reference panels of fibroblast and blood. We then evaluated the clinical performance of our outlier-based pipeline using positive samples with previously identified diagnostic findings from the Undiagnosed Diseases Network project. Our pipeline identified 19 of 20 positive findings in both fibroblast and blood samples. Our study provides a paradigm and necessary resources for independent laboratories to validate a clinical RNA-seq test.