Long-read sequencing with targeted assembly of the opsin locus accurately evaluates genes in expressed positions

The human opsin gene cluster at Xq28 contains highly similar OPN1LW and OPN1MW genes essential for red-green color vision. Current molecular methods cannot accurately analyze this complex locus, limiting diagnosis of color vision deficiencies (CVD) and detection of carrier status. We performed Nanopore long-read sequencing of 206 individuals, comparing alignment-based analysis with targeted de novo assembly. Alignment-based methods performed poorly, whereas targeted assembly achieved 99% concordance for OPN1LW and 92% for OPN1MW copy numbers and resolved gene order in all XY individuals and 87% of XX individuals. This approach detected CVD in 3.2% of XY individuals and identified 8% of XX individuals as carriers, consistent with population estimates. Moreover, it molecularly explained the phenotypic severity in a family with Bornholm eye disease and clarified carrier status in an XX individual suspected of carrying two CVD haplotypes. Our approach provides a comprehensive, reference-free method for accurate analysis of expressed opsin genes and reliable CVD carrier detection.