Nanopore Deep Sequencing as a Tool to Characterize and Quantify Aberrant Splicing Caused by Variants in Inherited...

Area of Science:

• Genomics
• Molecular Diagnostics
• Bioinformatics

Background:

• Splicing variants contribute to inherited diseases, but their diagnostic impact is underestimated due to prediction and detection challenges.
• Current molecular diagnostic methods struggle to identify and quantify all aberrant splicing events, especially low-abundance transcripts.

Purpose of the Study:

• To evaluate Nanopore sequencing for characterizing and quantifying aberrant splicing events caused by candidate variants.
• To assess the utility of Nanopore sequencing in diagnosing inherited retinal dystrophies.

Main Methods:

• In silico prediction of splicing variant effects.
• Functional characterization using minigene assays or whole blood cDNA.
• Nanopore sequencing of PCR-amplified cDNA to identify and quantify full-length transcripts.

Main Results:

• Thirteen of nineteen candidate variants (68%) induced aberrant splicing, including cryptic splice site activation, exon skipping, and pseudoexon inclusion.
• Nanopore sequencing accurately identified and quantified aberrant transcripts, aligning with in silico predictions.
• The method successfully detected low-abundance transcripts missed by conventional RT-PCR and Sanger sequencing.

Conclusions:

• Nanopore sequencing is a powerful tool for comprehensive analysis of aberrant splicing events in inherited diseases.
• This technology enhances molecular diagnostics by reliably detecting and quantifying splice variants, including those previously undetectable.