Development of a Versatile, Near Full Genome Amplification and Sequencing Approach for a Broad Variety of HIV-1 Group M Variants

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.

Keywords: HIV-1 group M subtype-independent approach; Near full genome amplification and sequencing; bulk sequencing and cloning; rational primer design; single-genome amplification (SGA); third-generation sequencing (TGS).

Figure 1

Near full genome sequence amplification approaches. (A) Genome map of HIV-1 reference strain HxB2 (GenBank: K03455). (B) One-amplicon approach to obtain a single 9.1 kb PCR fragment (near full genome) shown in green. (C) Two-amplicon approach with two overlapping PCR fragments (shown in red), with half genome 1 (HG1) 5.2 kb and half genome 2 (HG2) 4.6 kb in size. (D) Multiple-amplicon approach: Selection of eight overlapping PCR fragments (shown in black), i.e. gag (1.5 kb), gagpol (4.5 kb), pol (3.2 kb), vifvpu (1.5 kb), vif/gp120 (3 kb), env (3.4 kb), gp120 (1.7 kb) and gp41/nef (1.8 kb). Positions of primers are schematically shown with gray arrows in (A) and (B).

Figure 2

Conserved primer binding sites for HIV-1 near full genome amplification. Multiple sequence alignments of primer binding sites across ten different pure (sub)-subtypes and CRFs, as relevant for the near full genome amplification approach 1. For each (sub)-subtype/CRF, four reference sequences were selected that broadly cover the respective clade; for the binding site of primer 3’UTR rev 1, only two A2 and F2 reference sequences were available from the LANL database. The numbering of primer binding regions is based on the HxB2 reference strain (GenBank: K03455). Positions (pos) of primer binding sites are shown in brackets (HxB2 pos. 596–9542). Orange and green arrows along the HxB2 genome map indicate first round and second round primer positions, respectively. Black dots and colored letters indicate matches and mismatches with primer nucleotides shown on top, respectively.

Figure 3

Gel electrophoresis of PCR products of different lengths for composite near full genome analysis. (A) HIV-1 near full genome (NFG; green) and half genome (HG; red) amplicons obtained with different primer sets. (B) Diverse amplicons within half genome 1 (HG1). (C) Diverse amplicons within half genome 2 (HG2). Amplicons were separated on an ethidium bromide-stained 0.8% agarose gel. MassRuler DNA ladder was used for amplicon size determination. The amplicons shown in the gels were obtained from the following samples: NYU6541-6 (NFG1), NYU129-5 (NFG2), LB089-1 (NFG3 and env), NYU1122-1 (NFG4, HG1, and HG1a), NYU119-3 (HG2, pol, and gag), NYU1999 (gagpol), NYU6556-3 (HG1b), BDHS33 (vif/gp120), NYU124-2 (gp120), and MDC131-1 (gp41/nef and vifvpu).

Figure 4

Development of a near full genome sequencing approach using recombinant and pure subtype strains. Genome maps of four HIV-1 strains, which were used to develop the near full genome sequencing approach. The determined subtypes are indicated using the recombinant drawing tool of the LANL database. NYU6541_6, NYU6506_8, LB002_1 and strain 0526 correspond to a unique recombinant form (URF), the circulating recombinant form CRF02_AG, sub-subtype F2, and subtype B, respectively.

Figure 5

Comparative recombinant breakpoint analysis of near full genome sequences obtained with four different amplification, sequencing, and cloning strategies. Using sample NYU6541_6, different amplification, sequencing, and cloning techniques were studied: (A) third-generation sequencing (TGS), (B) bulk amplification and Sanger sequencing, (C) cloning after bulk amplification, followed by Sanger sequencing, and (D) single genome amplification (SGA) and Sanger sequencing. BootScan plots (Simplot software) are shown for near full genome sequences of NYU6541_6 with window size 200 and step size 20. Breakpoint patterns were determined by analyzing the query sequence against CRF02_AG (red), F2 (cyan), C (dark gray), and B (light gray) reference strains. The genomic regions gag, pol, vif/vpr/vpu and env are schematically shown along the x-axis in green, red, yellow, and purple bars, respectively. The y-axis indicates bootstrap values obtained after 250 replicative measurements.