Next-generation sequencing of HIV-1 single genome amplicons

Abstract

The analysis of HIV-1 sequences has helped understand the viral molecular epidemiology, monitor the development of antiretroviral drug resistance, and design candidate vaccines. The introduction of single genome amplification (SGA) has been a major advancement in the field, allowing for the characterization of multiple sequences per patient while preserving linkage among polymorphisms in the same viral genome copy. Sequencing of SGA amplicons is performed by capillary Sanger sequencing, which presents low throughput, requires a high amount of template, and is highly sensitive to template/primer mismatching. In order to meet the increasing demand for HIV-1 SGA amplicon sequencing, we have developed a platform based on benchtop next-generation sequencing (NGS) (IonTorrent) accompanied by a bioinformatics pipeline capable of running on computer resources commonly available at research laboratories. During assay validation, the NGS-based sequencing of 10 HIV-1 env SGA amplicons was fully concordant with Sanger sequencing. The field test was conducted on plasma samples from 10 US Navy and Marine service members with recent HIV-1 infection (sampling interval: 2005–2010; plasma viral load: 5,884–194,984 copies/ml). The NGS analysis of 101 SGA amplicons (median: 10 amplicons/individual) showed within-individual viral sequence profiles expected in individuals at this disease stage, including individuals with highly homogeneous quasispecies, individuals with two highly homogeneous viral lineages, and individuals with heterogeneous viral populations. In a scalability assessment using the Ion Chef automated system, 41/43 tested env SGA amplicons (95%) multiplexed on a single Ion 318 chip showed consistent gene-wide coverage >50×. With lower sample requirements and higher throughput, this approach is suitable to support the increasing demand for high-quality and cost-effective HIV-1 sequences in fields such as molecular epidemiology, and development of preventive and therapeutic strategies.

Evolution of Multiple Domains of the HIV-1 Envelope Glycoprotein during Coreceptor Switch with CCR5 Antagonist Therapy

“…Single-genome amplification of subgenomic and full-length genomic HIV-1 sequences was developed to preserve linkage among polymorphisms in the same viral genome copy, to limit impact from PCR-induced misincorporation and recombination or bacterial selection during cloning. Although most studies have used standard Sanger sequencing for analysis, our previous study and a couple of others sequenced Illumina- or PacBio-based pools of single-genome amplicons ( 33 , 44 – 46 ). Recently, Pacific Biosciences SMRT and Oxford MinION Nanopore sequencing were applied for longer reads ( 47 ).…”

Evidence of recurrent selection of mutations commonly found in SARS-CoV-2 variants of concern in viruses infecting immunocompromised patients

“…Single genome amplification (SGA) was performed with the sample collected on June 9th (day 22) ( Figure 1B ) to confirm the results found by full-length genome next generation sequencing. The SGA protocol was adapted from Kijak et al (2019) using nested PCR with primers 35L/39R (first round) and 36L/38R (second round) for nsp6 amplification and primers 72L/74R (first round) and 72L/73R (second round) to amplify a fragment of spike gene. Primers were selected from the ARTIC network n-Cov-2019 V.3 primer set.…”

So Pathogenic or So What?—A Brief Overview of SIV Pathogenesis with an Emphasis on Cure Research

“…Unfortunately, reducing the time constraints and labor by using subgenomic sequencing introduces detection and accuracy problems due to either defects in regions outside of the amplified region or deletions overlapping the amplified region [404]. Utilizing next-generation sequencing is one method to increase efficiency and cost effectiveness [405][406][407] and has higher sensitivity than Sanger sequencing, allowing for a better detection of mutations [408]. The recently developed intact proviral DNA assay (IPDA) is based on digital droplet PCR (ddPCR) multiplex technology [409].…”