Development of novel methods to exploit next gen sequencing for HIV

描述(由申请人提供)：下一代测序(NGS，深度测序)正在彻底改变序列信息的使用，以了解基因组和基因组的可变性。虽然NGS允许对大基因组进行快速测序，但它也允许对具有小基因组的生物的遗传多样性进行分析。然而，当每个序列被认为是一个独立的观察/基因组时，使用NGS来测量种群多样性充满了伪影。测序方案的高错误率和在聚合酶链式反应扩增过程中的误掺引入了人工多样性。扩增后对相同模板序列的PCR重新采样引入了人工同质性。在将NGS应用于艾滋病毒-1等复杂人群的大多数情况下，这两个严重问题都没有得到解决。我们专门开发了Primer ID方法来克服这些问题。在本申请中，我们建议定义Primer ID策略的优点和缺点，并使用该策略来探索HIV-1种群结构和进化的基本特征。这些研究将为长期存在的重要问题提供明确的答案，这些问题影响到药物治疗之前预先存在的变异的相关性，以及艾滋病毒-1进化的潜在途径。这些实验将使用人类样本，因此结果将直接适用于在这种病原性感染的背景下理解病毒-宿主相互作用的性质。

DESCRIPTION (provided by applicant): Next Generation Sequencing (NGS, deep sequencing) is revolutionizing the use of sequence information to understand genomes and genome variability. While NGS has allowed the rapid sequencing of large genomes, it has also allowed the allowed an analysis of the genetic diversity in organisms with small genomes. However, the use of NGS to measure population diversity is fraught with artifacts in when each sequence is considered as an independent observation/genome. The high error rate of the sequencing protocols and misincorporation during PCR amplification introduce artifactual diversity. PCR resampling of the same template sequences after amplification introduces artifactual homogeneity. Neither of these serious problems is addressed in most of the application of NGS to complex populations such as HIV-1. We have developed the Primer ID approach specifically to overcome these problems. In this application we propose to define the strengths and weaknesses of the Primer ID strategy and to use this strategy to explore fundamental features of HIV-1 population structure and evolution. These studies will provide definitive answers to longstanding and important questions that impact the relevance of pre-existing variants prior to drug therapy and the potential pathways for HIV-1 evolution. The experiments will use human samples so that the results will be directly applicable to understanding the nature of virus-host interactions in the context of this pathogenic infection.