Mendelian disease affects approximately 1 in 17 people across the globe and has so far been associated with over 4800 genes. There is a compelling need to improve our understanding of rare genomic variation; current genetic analysis techniques for Mendelian disease have a limited success rate meaning that despite comprehensive genomic testing, many patients and families are left without a molecular diagnosis. It is a pivotal time for rare disease research, diagnostics and genomic medicine, with whole genome sequencing (WGS) likely to be the method of choice for genetic analysis for the short to mid-term future. However, there are significant limitations with the current chemistry and informatics technology, and our understanding/interpretation of variants; Next Generation Sequencing (NGS) relies on short, paired-end read sequencing of up to 150bp which comprise limited genomic context, and to truly capitalize on WGS coverage, we must better understand the variation outside of gene panels and coding exons that remain the focus of clinical diagnostics. Thus, there are many contributors to the missing heritability including undiscovered genes, variants of uncertain significance (VUS), non-exonic variants and structural rearrangements and genes/variants intractable to NGS pipelines. The primary goal of this study is to better understand missing heritability in rare disease. Previous work in my laboratory has demonstrated that novel genes and non-coding variants in known inherited retinal dystrophy (IRD) genes are a significant cause of disease and we have established methodology to be able to characterize the effect of splice variants using blood derived mRNA and nanopore sequencing. Simultaneous work on the utility of ultralong-read sequencing for difficult to resolve cases has shown promising preliminary results. This application aims to build on these findings and develop the studies in IRD genes, expanding to broader genetic disease patients and families who have undergone testing at GGC. Aim 1 will utilize existing anonymized WGS datasets from the 100,000 genomes project to identify novel candidate disease genes and pathogenic noncoding variants by applying cutting-edge bioinformatics tools. Up to two genes will be taken forward in functional studies in collaboration with other research groups as separate projects. Noncoding/VUS will be analyzed by RT-PCR/nanopore sequencing to determine damaging effects and thus reclassify those variants as pathogenic. Aim 2 will investigate the potential utility of adaptive sampling targeted nanopore sequencing for clinical use in unsolved patients and families, and those where it is suspected that the culprit gene is intractable to NGS. For this exploratory work, we will use the model of IRD to test this in the first instance, targeting genes including OPN1LW/OPN1MW, ABCA4, USH2A, EYS, PRPF31, TYR, genes known to have limitations with coverage, phasing or haplotypes that limit the ab...