Project Summary Long-read sequencing in the form of Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT) have upended the preconceived notion of the capabilities of DNA sequencing applications. However, the molecular biology and computational methods available for long-read sequencing still lag behind the rich ecosystem of methods available for short- read technology in terms of both capability and usability. Because of this, the potential of long-read sequencing methods to benefit biomedical research remains largely unrealized. To address this shortfall, my lab will continue our work on developing and using methods that push the capabilities of long-read sequencing technology. First, we will generate complete isoform-level tissue and cell- type transcriptomes for human and mouse which will be invaluable to the biomedical research community investigating gene and isoform expression using RNA-seq. In addition, access to these transcriptomes will strongly benefit assays that rely on prior knowledge of which isoforms are expressed at what level in any particular cell-type or tissue. Second, we will develop an easy-to-use, ultra-accurate, and read-length agnostic sequencing method which will democratize the use of high-throughput sequencing technology and thereby increase the diversity of the genomics workforce by enabling a much larger number of less well funded labs to perform high-quality high-throughput DNA sequencing assays.