Near-Full-Length Single-Genome HIV-1 DNA Sequencing.
Near-Full-Length Single-Genome HIV-1 DNA Sequencing.
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DOI：
10.1007/978-1-0716-1871-4_23
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发表时间：
2022
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HIV-1整合到人类染色体中，建立病毒感染细胞的终身储存库。然而，大多数整合的病毒DNA显示出致命的缺陷，可能是由于在病毒RNA的逆转录过程中引入的错误。识别和定量HIV-1 DNA序列是基因组完整的，可以引起反弹的病毒血症在抗逆转录病毒治疗中断是理解HIV-1储库细胞的复杂性和进化动力学的关键步骤。在这里，我们描述了FLIP-Seq（全长个体前病毒测序），一种用于分析人类细胞中HIV-1 DNA的近全长、单基因组下一代测序方法。简言之，该技术涉及将前病毒DNA连续稀释成单个基因组，扩增近全长病毒DNA，扩增产物的深度测序，以及设计用于区分基因组完整的HIV-1 DNA与缺陷病毒DNA种类的生物计算分析。该程序可以用来自高度纯化的CD 4 T细胞亚群的少量细胞进行，允许产生给定细胞群体中存在的病毒序列的绝对定量，提供对完整前病毒的系统发生关联的洞察，并且可以鉴定可能来源于克隆扩增的储库细胞的序列相同的前病毒的比例。
HIV-1 integrates into human chromosomes to establish a lifelong reservoir of virally infected cells. However, the majority of integrated viral DNA shows lethal defects, likely due to errors introduced during reverse transcription of viral RNA. Identifying and quantifying HIV-1 DNA sequences that are genome-intact and can give rise to rebound viremia during antiretroviral treatment interruption are critical steps for understanding the complexity and evolutionary dynamics of HIV-1 reservoir cells. Here, we describe FLIP-Seq, (Full-Length Individual Proviral Sequencing) a near full-length, single-genome next-generation sequencing approach for analyzing HIV-1 DNA in human cells. Briefly, this technique involves sequential dilution of proviral DNA to single genomes, amplification of near full-length viral DNA, deep sequencing of amplification products, and a biocomputational analysis designed to distinguish genome-intact HIV-1 DNA from defective viral DNA species. This procedure can be performed with small numbers of cells from highly purified CD4 T cell subsets, allows to generate an absolute quantification of viral sequences present in a given cell population, provides insight into phylogenetic associations of intact proviruses, and can identify proportions of sequence-identical proviruses likely derived from clonally expanded reservoir cells.