Project Summary / Abstract The equipment supplement seeks to acquire a PromethION 24 long read sequencer to increase the impact of the award grant. The parent proposal presented a pilot screen of more than 32K variants from myCode and ClinVar that suggest 1-2% of exonic mutations affect splicing. The pilot study also revealed that splicing mutations are not uniformly distributed across disease genes or even within genes. This proposal will continue this effort on variants from UK biobank, AllofUs, ClinVar and GTEx that localize to actionable genes. Splicing mutations do not occur uniformly across exons. The proposal seeks to map susceptibility to splicing mutations and identify hotspot exons (exons unusually susceptible to splicing mutations) in the genome. The long read sequencer will enable full isoform analysis which will discover complex splicing events that contain multiple exons. In addition to identifying loss of splicing variants, the little studied problem of gain of splicing variants (i.e. single base mutations that can activate pseudoexons) will also be explored. Preliminary studies locate many intronic regions in pre-mRNA that contain all-but-one of the numerous cis-elements necessary for splicing. In addition to mapping susceptibility to splicing mutations, these efforts will contribute to the basic science of combinatorial signal recognition with a high-level approach that will define mechanisms of splice site selection. Finally, we present a principled method of restoring splicing to mutated exons using antisense oligonucleotides (ASO) that weaken flanking splice sites. Here again long read sequencing will be used to study splicing in its native state and properly identify complex events.