HiFi long-read RNA sequencing enhances clinical diagnostics in rare disorders
Jaramillo Oquendo, Carolina Ferraro, Federico Wai, Htoo A Ferrao, Heather van der Linde, Herma Karelioti, Evita Tseng, Liz Dhillon, Harsharan Holt, Sam Bunyan, David J
Jaramillo Oquendo, Carolina
Ferraro, Federico
Wai, Htoo A
Ferrao, Heather
van der Linde, Herma
Karelioti, Evita
Tseng, Liz
Dhillon, Harsharan
Holt, Sam
Bunyan, David J
Abstract
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. We evaluated the potential of PacBio long-read RNA-seq to detect pathogenic splicing events in rare disorders, comparing its performance to short-read RNA-seq. Participants from the UK (n = 23) and the Netherlands (n = 2) with suspected splice-altering variants underwent long-read RNA-seq following the Kinnex full-length RNA protocol. HiFi reads from the Revio instrument were processed using the Read Segmentation and Iso-Seq workflow and then classified and filtered using Pigeon. Detection of disease genes 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 globin depletion in blood. Transcript abundance estimates showed strong concordance between short- and long-read platforms (Pearson r = 0.86 and 0.61 in blood and fibroblasts, respectively). LRS captured 21 confirmed known events, and revealed additional transcript-level effects in eight cases. This included intron retention, multiple exon skipping, leaky splicing, variant phasing, and isoform switching. These results demonstrate that long-read RNA-seq enhances detection and interpretation of clinically relevant splicing events, supporting its integration into diagnostic workflows for rare diseases.
MIDER Authors
Date
2026-03-10
Type
Article
Subject
Rare diseases, RNA, Genes, Fibroblasts
Collections
Citation
Jaramillo Oquendo C, Ferraro F, Wai HA, Ferrao H, van der Linde H, Karelioti E, Tseng L, Dhillon H, Holt S, Bunyan DJ, Donker Kaat L, van Dooren M, Zhou J, Ennis S, Holloway JW, van Ham TJ, Baralle D. HiFi long-read RNA sequencing enhances clinical diagnostics in rare disorders. Eur J Hum Genet. 2026 Mar 10. doi: 10.1038/s41431-026-02042-9. Epub ahead of print.
Journal / Source Title
European Journal of Human Genetics
DOI
10.1038/s41431-026-02042-9
PMID
41807732
Publisher
Nature Publishing Group
Publisher’s URL
https://www.nature.com/ejhg/