AUTHOR=Rodilla Cristina , Núñez-Moreno Gonzalo , Benitez Yolanda , Romero Raquel , Fernández-Caballero Lidia , Mínguez Pablo , Corton Marta , Ayuso Carmen TITLE=Cas9-targeted-based long-read sequencing for genetic screening of RPE65 locus JOURNAL=Frontiers in Genetics VOLUME=15 YEAR=2024 URL=https://www.frontiersin.org/journals/genetics/articles/10.3389/fgene.2024.1439153 DOI=10.3389/fgene.2024.1439153 ISSN=1664-8021 ABSTRACT=Introduction

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.

Methods

LRS was conducted in five inherited retinal dystrophy (IRD) patients harboring a monoallelic variant in RPE65 that remained uncharacterized after clinical exome sequencing (CES). CRISPR-Cas9 guide RNA probes were designed to target a 31 kb region, including the entire RPE65 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.

Results

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 RPE65 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.

Discussion

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.