Development of a Versatile, Near Full Genome Amplification and Sequencing Approach for a Broad Variety of HIV-1 Group M Variants
Development of a Versatile, Near Full Genome Amplification and Sequencing Approach for a Broad Variety of HIV-1 Group M Variants
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DOI：
10.3390/v11040317
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发表时间：
2019-04-01
期刊：
影响因子：
4.7
通讯作者：
Duerr, Ralf
中科院分区：
文献类型：
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作者：
Banin, Andrew N.;Tuen, Michael;Duerr, Ralf
为了全面评估HIV-1毒株的遗传组成，需要对HIV-1进行近全基因组测序。在人群范围内，它可以确定HIV-1的异质性和新/重组菌株的出现，同时对每个人来说，它构成了一种诊断工具，有助于针对病毒成分采取有针对性的治疗措施。对于各种HIV-1亚型的高效NFGS，仍然缺乏可靠和适应性强的技术。采用合理的引物设计，开发了一套广泛的引物，用于从血浆中扩增和测序多种HIV-1 M组变异。利用纯亚型和多种独特的重组形式（URF），开发了包含所有功能基因的NFGS的可变扩增子方法。成功鉴定出由A （A1）、B、F （F2）、G、CRF01_AE、CRF02_AG和CRF22_01A1亚型组成的23个不同基因组。无论病毒载量（306拷贝/mL）和扩增方法如何，NFGS方法都是稳健的。第三代测序（TGS）、单基因组扩增（SGA）、克隆和批量测序在亚型组成和重组断点模式方面得出了相似的结果。引入一种简单而通用的近全基因组扩增、测序和克隆方法，可以广泛应用于各种HIV-1亚型的系统发育研究，并有助于个性化HIV治疗和诊断。
Near full genome sequencing (NFGS) of HIV-1 is required to assess the genetic composition of HIV-1 strains comprehensively. Population-wide, it enables a determination of the heterogeneity of HIV-1 and the emergence of novel/recombinant strains, while for each individual it constitutes a diagnostic instrument to assist targeted therapeutic measures against viral components. There is still a lack of robust and adaptable techniques for efficient NFGS from miscellaneous HIV-1 subtypes. Using rational primer design, a broad primer set was developed for the amplification and sequencing of diverse HIV-1 group M variants from plasma. Using pure subtypes as well as diverse, unique recombinant forms (URF), variable amplicon approaches were developed for NFGS comprising all functional genes. Twenty-three different genomes composed of subtypes A (A1), B, F (F2), G, CRF01_AE, CRF02_AG, and CRF22_01A1 were successfully determined. The NFGS approach was robust irrespective of viral loads (306 copies/mL) and amplification method. Third-generation sequencing (TGS), single genome amplification (SGA), cloning, and bulk sequencing yielded similar outcomes concerning subtype composition and recombinant breakpoint patterns. The introduction of a simple and versatile near full genome amplification, sequencing, and cloning method enables broad application in phylogenetic studies of diverse HIV-1 subtypes and can contribute to personalized HIV therapy and diagnosis.