Improved Detection of Rare HIV-1 Variants using 454 Pyrosequencing

Abstract

454 pyrosequencing, a massively parallel sequencing (MPS) technology, is often used to study HIV genetic variation. However, the substantial mismatch error rate of the PCR required to prepare HIV-containing samples for pyrosequencing has limited the detection of rare variants within viral populations to those present above ~1%. To improve detection of rare variants, we varied PCR enzymes and conditions to identify those that combined high sensitivity with a low error rate. Substitution errors were found to vary up to 3-fold between the different enzymes tested. The sensitivity of each enzyme, which impacts the number of templates amplified for pyrosequencing, was shown to vary, although not consistently across genes and different samples. We also describe an amplicon-based method to improve the consistency of read coverage over stretches of the HIV-1 genome. Twenty-two primers were designed to amplify 11 overlapping amplicons in the HIV-1 clade B gag-pol and env gp120 coding regions to encompass 4.7 kb of the viral genome per sample at sensitivities as low as 0.01-0.2%.

Targeted deep sequencing of HIV-1 using the IonTorrentPGM platform

“…Following the principles stated by Larsen et al (2013) endpoint dilution employing the same amplification system was used for library preparation in order the estimate the number of amplifiable template copies present in the cDNA sample. This method was preferred rather than directly using pVL values from clinical testing, as the latter can underestimate the presence of amplifiable templates in the sample.…”

“…When applied to HIV-1, the measured noise of NGS results from the composite of: 1) reverse transcription (when using viral RNA (vRNA) as initial template), 2) PCR for library preparation, 3) emulsion PCR (ePCR) or cluster generation, and 4) sequencing. The introduction of noise in the first two processes can be limited using high-fidelity enzymes (Di Giallonardo et al, 2013; Larsen et al, 2013), whereas the end-user has little capacity to influence the conditions of processes 3 and 4. In the study of HIV-1, the capacity to accurately discern true genetic signals from background noise is paramount; especially considering that SNPs can drastically impact susceptibility to antiretroviral drugs (Richman et al, 1994) or immune effectors (Leslie et al, 2004).…”

Emerging Technologies for the Clinical Microbiology Laboratory

“…This is again in comparison to routine Sanger methods, which demonstrate a limit of detection of approximately 20 to 35% of the population (171). Accurate sequence data with error rates of Ͻ0.05% are easily achieved due to the high number of parallel reads, which provide highly redundant coverage of the target sequence (168,169). Pretherapy resistance testing has been recommended to identify quasispecies with mutations known to confer resistance to antiretrovirals and is also recommended following a rise in HIV load attributed to therapy failure (172).…”

“…For example, ultradeep sequencing has been successfully used to detect HIV quasispecies and the emergence of resistant subpopulations. Analysis of blood samples from HIV-infected patients using pyrosequencing identified strains with mutations in the viral reverse transcriptase gene at levels of Ͻ0.1% of the total viral population (168,169). Similarly, Ion Torrent sequencing was utilized to identify the emergence of mutations conferring resistance to nonnucleoside reverse transcriptase inhibitors (NNRTIs) and protease inhibitors (PIs) at a level of Ͻ1% of the total population through an average of 13,700ϫ coverage of the gag-pol loci, though it was noted that coverage decreased significantly in a homopolymeric region containing five consecutive guanine residues (170).…”

Fitness-Balanced Escape Determines Resolution of Dynamic Founder Virus Escape Processes in HIV-1 Infection

“…HIV RNA derived from plasma from seroconverting partners (SPs) were first sequenced a median of 25 days post onset of symptoms (dps) and then followed for a median of 827 dps, whereas transmitting partner (TP) plasma RNA was sequenced at one time point at a median of 28.5 dps in the SP (Table 1). Viral sequences corresponding to Gag p17 and p24 coding regions were obtained by 454 pyrosequencing of a median of 245 viral templates or by generating 15 to 20 single-template derived Sanger sequences when material and/or viral load was limiting (40). Phylogenetic analysis supported a single viral variant establishing infection in 5/6 SPs, whereas PIC51861 had at least two founding variants.…”