Can Nanopore sequencing reveal gene-splicing problems that conventional tests miss in inherited retinal disorders?

Details

Splicing variants are reported to account for less than 10% of molecular diagnoses of inherited diseases. The abstract suggests this is likely an underestimate: their effects can be difficult to predict, functional assays are needed, and some variants cannot be detected depending on their location and the sequencing technology used.

This laboratory study assessed the utility of Nanopore sequencing for characterizing and quantifying abnormal splicing events. Researchers selected 19 candidate splicing variants identified in patients with inherited retinal dystrophies. They used several in silico tools to predict the type and estimate the extent of variant-induced abnormal splicing.

The variants were functionally characterized using a minigene assay or whole blood-derived cDNA. To identify the resulting transcripts, the researchers combined PCR amplification of minigene-specific cDNA or the target gene in blood cDNA with Nanopore sequencing.

Thirteen out of 19 variants caused abnormal splicing, including activation of cryptic splice sites, exon skipping, pseudoexon inclusion, or combinations of these events. Nanopore sequencing identified full-length transcripts and allowed their precise quantification; these findings often agreed with the in silico predictions. The method reliably detected low-abundance transcripts that would not be detected by conventional strategies, such as RT-PCR followed by Sanger sequencing.