Novel Methods for Cell-free RNA Biomarker Characterization Using Nanopore Sequencing

Cancer is often detected as late-stage disease due to limitations in current detection methods. Existing diagnostic approaches can only identify morphological changes or lack the sensitivity required for early-stage detection. RNA liquid biopsies enable the comprehensive characterization of dynamic transcriptional changes resulting from the continuous release of cell-free RNA by tumor cells. I describe novel approaches for combining short and long-read sequencing for transcriptome assembly followed by quantification to analyze cell-free RNA in vitro and in vivo. I will present data applying this method to human lung cells transduced with mutant KRAS and treated with cigarette smoke extract to discover novel cell-free RNA biomarkers for lung cancer early detection. I will also discuss applying this method to assemble a cell-free transcriptome followed by short-read quantification for esophageal cancer early detection using patient plasma. Furthermore, I present novel approaches for discovering additional features of cell-free RNA with potential diagnostic utility. Finally, I present a novel computational approach for profiling non-human RNAs that may derive from the tumor microbiome. Collectively, my thesis work encompasses the development of novel experimental and computational methodologies to profile cell-free RNA biomarkers for cancer early detection.