Cas9-targeted-based long-read sequencing for genetic screening of <i>RPE65</i> locus

<h4>Introduction</h4>Long-read sequencing (LRS) enables accurate structural variant detection and variant phasing. When a molecular diagnosis is suspected, target enrichment can reduce the cost and duration of sequencing.<h4>Methods</h4>LRS was conducted in five inherited retinal dystrophy (IRD) patients harboring a monoallelic variant in <i>RPE65</i> that remained uncharacterized after clinical exome sequencing (CES). CRISPR-Cas9 guide RNA probes were designed to target a 31 kb region, including the entire <i>RPE65</i> locus. The DNA was sequenced on a MinION platform. Short-read ×30 whole-genome sequencing (WGS) was performed for five patients to validate nanopore results.<h4>Results</h4>The nanopore sequencing process yielded a median of 271 reads within the targeted region, with a mean depth of 109 and a median read size of 8 kb. All variants identified by CES have been detected using this approach, and no additional <i>RPE65</i> gene causative variants were found. Nanopore variant detection demonstrated performance akin to short-read WGS at similar coverage levels, although exhibiting increased false positive calls at lower coverage.<h4>Discussion</h4>In this study, we explore the advantages of using a targeted approach together with long-read sequencing to identify variants associated with IRD. The results underscore the utility of targeted long reads for characterizing patients affected by rare diseases when first-tier diagnostic tests are non-conclusive.