PROJECT SUMMARY/ABSTRACT Clinical genetic testing, including exome sequencing, has diagnosed many rare and novel genetic disorders. These diagnoses have helped guide treatment, suggest novel or precision therapies, and provide long-term prognostic information for individuals and their families. However, at least 50% of individuals with a suspected genetic disorder remain undiagnosed after a complete clinical evaluation, which can take years to complete. There are two major technical reasons for this low solve rate. First, structural variants such as large deletions, insertions, inversions, and repeat expansions account for a significant fraction of unsolved cases, but they are challenging to analyze using traditional short-read sequencing technologies. Second, many variants lie in recently duplicated genomic regions that are difficult to evaluate using short-read sequencing technologies. Recently, it has become economically feasible to pursue whole-genome long-read sequencing (LRS) of human genomes with sufficient coverage to analyze these complex genomic regions. This presents an opportunity to systematically evaluate the incremental diagnostic rate of LRS over current testing approaches and apply LRS technology to a large cohort of individuals with unsolved genetic disorders. Moreover, LRS is unique in that as a single test it has the potential to replace nearly all existing clinical genetic testing (microarray, exome, methylation studies), making it possible to perform a complete clinical genetic evaluation in days to weeks using a single dataset. The overall goal of this project is to establish a framework for the clinical adoption of long-read DNA and RNA sequencing by systematic evaluation of individuals with suspected or unsolved genetic disorders. To achieve this goal, LRS will be performed on 50 critically ill neonates and their parents who had nondiagnostic trio short-read whole-genome sequencing (WGS) and 25 trios from the same cohort who had diagnostic WGS (Aim 1). This will help establish the incremental diagnostic rate of LRS over short-read WGS. To evaluate the technical advantages of LRS over traditional clinical testing, I will sequence 50 individuals with a suspected genetic disorder who remain unsolved after at least clinical exome sequencing (Aim 2). In up to 20 persons that remain undiagnosed after LRS, long-read RNA sequencing will be used to identify high-priority regions that influence gene expression or splicing for further analysis. I will then evaluate the sensitivity of LRS as a single genetic test by sequencing 50 individuals undergoing a typical outpatient clinical evaluation (Aim 3). Demonstrating that LRS can be used as a single data source will help streamline the current stepwise approach to clinical testing, leading to an increase in the diagnostic rate and an overall cost savings to the health system through reduced testing and clinic visits. Overall, the aims outlined in this proposal will benefit patients and families by increasing the rate of genetic diagnosis, decreasing the time to diagnosis, and identifying novel disease-causing mechanisms, which will lead to a better understanding of the causes of human disease.

PROJECT NARRATIVE Although the widespread adoption of clinical exome sequencing has helped identify the genetic basis of many previously unsolved Mendelian disorders, approximately 50% of individuals remain unsolved after a complete clinical workup. Long-read sequencing has the potential to identify and resolve many complex or difficult-to- identify variants such as insertions, deletions, repeat expansions, and variants within duplicated genes, but it has yet to be systematically evaluated in a clinical cohort. Here I propose a set of experiments which seek to evaluate the incremental diagnostic rate of long-read sequencing in cohorts that have had extensive but nondiagnostic clinical genetic testing and to compare long-read sequencing as a single test to a standard outpatient clinical workup.