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

感染人类并导致疾病的病毒在体内以相关但不相同的遗传变异的准种存在。 这些准种可以通过个体中少量创始者变体的多样化而产生，这些变异是在病毒复制的进行中通过无害随机突变的逐渐积累而产生的。 由这些病毒变体编码的多样性为病毒提供了重要的持久性机制，因为宿主介导的抗病毒应答或治疗性施用的抗病毒干预可能无法对具有某些逃逸突变的变体起作用。 这可能会产生重要的医疗后果，例如与艾滋病毒耐药性相关的不良临床结果。 准确表征来自感染个体的样品中的病毒准种的遗传多样性需要对单个分子进行高通量测序，有时需要在数千个碱基对长度的区域上进行测序。 目前的高通量测序技术虽然非常强大，但在应对这一挑战的能力方面受到限制。 因此，我们正在开发新的湿实验室和生物信息学方法，用于病毒基因组的高通量单分子序列分析，并将其应用于患有医学重要病毒的人的样本，最初集中在HIV上。

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.