HiFi Long-Read RNA Sequencing Enhances Clinical Diagnostics in Rare Disorders

Splice-disrupting variants are estimated to account for one-third of disease-causing variants, yet many remain underrepresented in clinical databases due to limitations in detecting splicing changes beyond canonical splice sites. Short-read RNA sequencing (RNA-seq) has proved to be a valuable complement in clinical practice to address this gap, however, the added value of long-read RNA-seq is unclear. Here, we aimed to assess the clinical utility of PacBio long-read RNA-seq to characterise pathogenic aberrant splicing in rare disorders compared to short-read RNA-seq. Participants from the UK and the Netherlands with suspected splice-altering variants underwent long-read RNA-seq. 28 blood samples and four fibroblast cell lines were sequenced following the Kinnex full-length RNA protocol. Detection of disease genes (OMIM and PanelApp) was comparable with short reads, with fibroblast capturing more transcripts overall. Novel isoforms accounted for ~ 14% of detected transcripts in both tissues, increasing following cycloheximide treatment in fibroblasts and decreasing following goblin depletion in blood. Long-read RNA-seq detected events missed by short-reads including intron retention, multiple exon skipping, differential transcript usage, leaky splicing and variant phasing. In one case long reads revealed that a splice region variant in RPS7 skewed expression toward an unannotated intron 6-retained transcript, likely leading to protein deficiency explaining previous ambiguous results in patient with Diamond-Blackfan anaemia. In another case, we identified a retrotransposon-induced isoform switch in TCOF1 causing Treacher Collins syndrome. Both examples unresolved by short reads. Thereby long-read RNAseq has the potential to improve the detection of clinically relevant transcripts when used in a clinical setting.