Real-time Analysis for the Phylogenetic Investigation of Drug-resistant HIV clusters (RAPID-HIV)

项目摘要 该项目的总体目标是实现对HIV-1耐药性和传播的实时监测 集群使用集成的计算管道和新的扩增子的方法，我们已经开发 用于纳米孔测序这个项目很重要，因为全球范围内对ART耐药性的上升是 威胁到在抑制病毒方面取得的重大进展。在非洲，这种威胁尤其严重 其中几个国家报告说，治疗前艾滋病毒耐药性水平超过10%， 标准一线治疗便携式测序技术的出现意味着病毒基因组测序 现在，它可以对公共卫生应对耐药性激增产生更大的影响。在 本提案中，我们将使用MinION测序设备并评估其性能特征 （准确性，特异性，灵敏度）的一种新的基于扩增子的方法，以准确地表征 使用全长pol扩增子的HIV-1多样性。由于HIV-1的偏向性核苷酸组成， 基因组和原始纳米孔数据的复杂性，我们将确定我们如何能够提高原始读取 准确性，并简化非生物信息学专家对纳米孔测序数据的分析。基于 乌干达渔业社区中HIV-1风险升高，我们假设来自 艾滋病患者经常携带耐药性突变，并成为耐药性的储存库。因此，我们需要 了解HIV-1感染者的传播动力学和传播耐药性的演变 维多利亚湖周围的渔业社区。该提案将整合测序、碱基判定和 使用集成的工作流程进行近实时分析，以实现HIV-1耐药性监测， 传输集群的检测。为了实现这些目标，我们将使用一组HIV-1样本 包括局部循环菌株，以评价我们的扩增子的性能特征 使用全长pol扩增子准确表征HIV-1多样性的测序方法。我们将 开发针对HIV-1的定制训练碱基判定模型，以准确量化耐药性， 实施实时基因分型和耐药性报告的集成分析管道， 非生物信息学专家对纳米孔测序数据的分析。最后，我们将利用我们最近的 我们开发的扩增子测序方法与我们的实时分析管道相结合，以了解 HIV-1感染者体内的传播动力学和传播耐药性的演变 维多利亚湖周围的渔业社区。总之，这些结果将促进我们对 艾滋病毒-1在渔业社区中的传播动态，并评估 这一高风险亚群。

Project Abstract The overall aim of this project is to enable real-time surveillance of HIV-1 drug resistance and transmission clusters using an integrated computational pipeline and novel amplicon approach that we have developed for nanopore sequencing. This project is important because globally the rise of resistance to ART is threating the significant progress made in suppressing the virus. In Africa, this threat is particularly acute where several countries have reported levels of pre-treatment HIV resistance exceeding 10% to the standard first-line therapy. The advent of portable sequencing technology means viral genomic sequencing is now positioned to exert a greater impact on the public health response to the upsurge in resistance. In this proposal we will use the MinION sequencing device and assess the performance characteristics (accuracy, specificity, sensitivity) of a novel amplicon-based approach in order to accurately characterize HIV-1 diversity using full-length pol amplicons. Owing to the biased nucleotide composition of the HIV-1 genome and the complexity of the raw nanopore data we will identify how we can improve raw read accuracy and simplify the analysis of nanopore sequencing data by non-bioinformatics experts. Based on the elevated HIV-1 risks within Ugandan fishing communities we hypothesize that viral clusters from fisherfolk frequently harbor drug-resistance mutations and act as reservoirs of resistance. Thus, we need to understand the transmission dynamics and evolution of transmitted drug resistance within HIV-1 infected fishing communities around Lake Victoria. This proposal will integrate sequencing, basecalling, and analysis in near real-time using an integrated workflow to enable HIV-1 drug resistance surveillance and the detection of transmission clusters. To achieve these objectives, we will use a panel of HIV-1 samples comprising locally circulating strains in order to evaluate the performance characteristics of our amplicon sequencing approach to accurately characterize HIV-1 diversity using full-length pol amplicons. We will develop custom trained basecalling models specific to HIV-1 to accurately quantify drug resistance and implement an integrated analysis pipeline for real-time genotyping and drug resistance reporting to simplify the analysis of nanopore sequencing data by non-bioinformatics experts. Lastly, we will use our recently developed amplicon sequencing approach coupled with our real-time analysis pipeline to understand the transmission dynamics and evolution of transmitted drug resistance within HIV-1 infected individuals from fishing communities around Lake Victoria. Together, these results will advance our understanding of the transmission dynamics of HIV-1 among fishing communities and evaluate the burden of resistance among this high-risk sub-population.