High-throughput, single-molecule sequence analysis of virus populations in vivo

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Viruses that infect humans and cause disease exist in vivo as quasispecies of related but non-identical genetic variants. These quasispecies can arise by diversification of a small number of founder variants in an individual by the progressive accumulation of non-deleterious random mutations during ongoing rounds of virus replication. The diversity encoded by these virus variants provides the virus with an important mechanism of persistence, as host-mediated antiviral responses or therapeutically-administered antiviral interventions may fail to act against variants with certain escape mutations. This can have important medical consequences, as exemplified by poor clinical outcomes associated with drug-resistance in HIV. Accurately characterizing genetic diversity of virus quasispecies in samples from infected individuals requires high-throughput sequencing of single molecules, sometimes over regions thousands of base-pairs in length. Current technologies for high-throughput sequencing, though very powerful, are limited in their ability to address this challenge. We are thus developing novel wet-lab and bioinformatic methods for high-throughput, single-molecule sequence analysis of virus genomes, and applying them to samples from people with medically-important viruses, focused initially on HIV.