Third-Generation Sequencing (TGS) technology offers an exciting breakthrough opportunity for virology researchers. However, TGS faces technical challenges including a relatively high base calling error rate that is hard to distinguish from the high mutation rate of viruses. This project will develop novel tools for TGS by taking advantages of existing development focusing on base calling of TGS, viral evolution and viral transmission. The project will develop tools characterizing viral transmissions and viral evolution in the presence of sequencing errors and will validate the tools by analyzing HIV epidemiology, evolution, and pathogenesis. These tools will enhance our understanding of HIV and will be widely applicable to study other viral evolution, epidemiology, and pathogenesis questions.