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Abstract
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.
Highlights
HIV-1 full genome sequencing is a challenging task due to the broad degree of HIV-1 genomic diversity worldwide
Before sample collection, informed consent was obtained from the study participants, who were all part of a cohort of HIV positive individuals; this cohort is monitored at the Medical Diagnostic Center (MDC) in Yaoundé, Cameroon in collaboration with the
The protocol should be suitable for several downstream methods including high-throughput sequencing (NGS or Third-generation sequencing (TGS)), single genome amplification (SGA), cloning, and bulk sequencing, and applicable to modern practice in well-equipped labs as well as to basic procedures in resource-limited countries
Summary
HIV-1 full genome sequencing is a challenging task due to the broad degree of HIV-1 genomic diversity worldwide. HIV-1 group M has spread globally, causing more than 85% of global HIV infections and can be subdivided into nine subtypes (A–D, F–H, J, and K), at least six sub-subtypes of A and F (A1–A6, F1–F2), currently at least 98 circulating recombinant forms (CRFs) [5], and numerous unique recombinant forms (URFs) [6]. CRFs and URFs are composed of two or more (sub)-subtypes; while CRFs have been identified in at least three epidemiologically unlinked individuals, URFs are defined as recombinants without evidence of onward transmission [5]. Including sequences that remain unclassified or can be attributed to ancient strains [10] poses a significant challenge for HIV-1 molecular surveillance, diagnosis, and therapy [11,12]. Robust and versatile amplification and sequencing protocols are needed that apply to such strains over the near full genome (NFG)
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