HiFi long-read RNA sequencing enhances clinical diagnostics in rare disorders

Area of Science:

• Genomics
• Molecular Biology
• Bioinformatics

Background:

• Splice-disrupting variants contribute to one-third of genetic diseases but are often missed by current clinical detection methods.
• Short-read RNA sequencing (RNA-seq) aids in detecting splicing changes, but the utility of long-read RNA-seq remains unclear.

Purpose of the Study:

• To evaluate the effectiveness of PacBio long-read RNA sequencing (LRS) in identifying pathogenic splicing events in rare genetic disorders.
• To compare the performance of LRS against short-read RNA sequencing for detecting splicing alterations.

Main Methods:

• Long-read RNA sequencing was performed on participants with suspected splice-altering variants using the Kinnex full-length RNA protocol on the Revio instrument.
• HiFi reads were processed using Read Segmentation and Iso-Seq, followed by classification and filtering with Pigeon.
• Data analysis focused on detecting known and novel splicing events and comparing transcript abundance with short-read data.

Main Results:

• Long-read RNA sequencing detected disease-relevant splicing events comparable to short-read RNA sequencing, with fibroblasts yielding more transcripts.
• Novel isoforms constituted approximately 14% of detected transcripts, with variations observed based on tissue type and experimental treatment.
• Transcript abundance estimates showed high concordance between short-read and long-read platforms (Pearson r = 0.86 in blood, 0.61 in fibroblasts).
• LRS identified 21 known splicing events and revealed additional transcript-level effects in eight cases, including intron retention, exon skipping, and isoform switching.

Conclusions:

• Long-read RNA sequencing significantly improves the detection and interpretation of clinically relevant splicing events in rare diseases.
• The findings support the integration of LRS into diagnostic workflows for rare genetic disorders, enhancing diagnostic yield.