Abstract We propose to use third generation, long-read sequencing (LRS) to resolve inconclusive diagnoses and maximize the diagnostic yield in patients with suspected Mendelian disorders. The diagnostic yield of whole exome and genome sequencing stands at 40-50% including a significant proportion of patients receiving an inconclusive diagnosis. The limitations of the current short-read technologies can be attributed to the negative and inconclusive molecular diagnoses that prevent us from maximizing the diagnostic yield. Also, the vast majority of the patients undergo a long diagnostic odyssey due to the practice of sequential testing paradigm. In the proposed study, we will perform LRS in a cohort of patients with heterogenous pediatric disorders to resolve a) inconclusive diagnoses in patients with a single disease associated variant in an autosomal recessive disorder gene, and two disease associated variants in the same gene with unknown phase, and b) variants of uncertain significance or variant of unknown parent-of-origin in patients with epigenetic or imprinting disorder. We will determine the diagnostic utility of LRS and its impact on uncertainty in diagnoses against the current standard of care exome/ genome sequencing. We will build upon the existing resources of our clinical lab and our own cutting-edge genomic and bioinformatic capabilities. We anticipate that our studies will contribute new knowledge about the increased diagnostic yield from resolution of inconclusive diagnoses and lead to the faster adoption of long-reads in clinical settings.