Amplification of Near Full-length HIV-1 Proviruses for Next-Generation Sequencing
Amplification of Near Full-length HIV-1 Proviruses for Next-Generation Sequencing
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DOI：
10.3791/58016
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发表时间：
2018-10-01
影响因子：
1.2
通讯作者：
Palmer, Sarah
中科院分区：
文献类型：
--
作者：
Hiener, Bonnie;Eden, John-Sebastian;Palmer, Sarah
全长个体原病毒测序 (FLIPS) 测定是一种高效、高通量的方法，旨在对单一、接近全长（完整和有缺陷）的 HIV-1 原病毒进行扩增和测序。 FLIPS 可以确定细胞群内整合的 HIV-1 的遗传组成。通过识别逆转录过程中出现的 HIV-1 原病毒序列中的缺陷，例如大的内部缺失、有害终止密码子/超突变、移码突变以及病毒颗粒成熟所需的顺式作用元件的突变/缺失，FLIPS 可以识别无法复制的整合原病毒。 FLIPS 检测可用于识别缺乏这些缺陷并因此具有潜在复制能力的 HIV-1 原病毒。 FLIPS 方案包括：HIV-1 感染细胞的裂解、近全长 HIV-1 原病毒的巢式 PCR（使用针对 HIV-1 5' 和 3' LTR 的引物）、DNA 纯化和定量、下一代测序 (NGS) 的文库制备、NGS、原病毒重叠群的从头组装以及用于识别具有复制能力的原病毒的简单消除过程。 FLIPS 比用于对整合的 HIV-1 原病毒进行测序的传统方法（例如单原病毒测序）具有优势。 FLIPS 能够扩增并测序接近全长的原病毒，从而能够确定复制能力，并且还使用更少的扩增引物，从而防止引物错配的后果。 FLIPS 是了解整合型 HIV-1 原病毒遗传图谱的有用工具，尤其是在潜伏病毒库中，然而，它的利用可以扩展到需要整合型 HIV-1 遗传组成的任何应用。
The Full-Length Individual Proviral Sequencing (FLIPS) assay is an efficient and high-throughput method designed to amplify and sequence single, near full-length (intact and defective), HIV-1 proviruses. FLIPS allows determination of the genetic composition of integrated HIV-1 within a cell population. Through identifying defects within HIV-1 proviral sequences that arise during reverse transcription, such as large internal deletions, deleterious stop codons/hypermutation, frameshift mutations, and mutations/deletions in cis acting elements required for virion maturation, FLIPS can identify integrated proviruses incapable of replication. The FLIPS assay can be utilized to identify HIV-1 proviruses that lack these defects and are therefore potentially replication-competent. The FLIPS protocol involves: lysis of HIV-1 infected cells, nested PCR of near full-length HIV-1 proviruses (using primers targeted to the HIV-1 5' and 3' LTR), DNA purification and quantification, library preparation for Next-generation Sequencing (NGS), NGS, de novo assembly of proviral contigs, and a simple process of elimination for identifying replication-competent proviruses. FLIPS provides advantages over traditional methods designed to sequence integrated HIV-1 proviruses, such as single-proviral sequencing. FLIPS amplifies and sequences near full-length proviruses enabling replication competency to be determined, and also uses fewer amplification primers, preventing the consequences of primer mismatches. FLIPS is a useful tool for understanding the genetic landscape of integrated HIV-1 proviruses, especially within the latent reservoir, however, its utilization can extend to any application in which the genetic composition of integrated HIV-1 is required.