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形式存在。这些准种可能是通过在进行中的几轮病毒复制过程中通过逐渐积累非有害的随机突变而使个体中的少量创始人变体多样化而产生的。这些病毒变种编码的多样性为病毒提供了一个重要的持久性机制，因为宿主介导的抗病毒反应或治疗用的抗病毒干预可能无法对抗具有某些逃逸突变的变种。这可能会产生重要的医学后果，与艾滋病毒耐药性有关的不良临床结果就是例证。 准确描述感染个体样本中病毒准种的遗传多样性需要对单分子进行高通量测序，有时需要对数千个碱基对的区域进行高通量测序。目前的高通量测序技术虽然非常强大，但解决这一挑战的能力有限。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，并将它们应用于最初专注于HIV的具有重要医学意义的病毒携带者的样本。

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.