Comparison of ultra-deep versus Sanger sequencing detection of minority mutations on the HIV-1 drug resistance interpretations after virological failure

Mohamed, Sofiane; Pénaranda, Guillaume; Gonzalez, Dimitri; Camus, Claire; Khiri, Hacène; Boulmé, Ronan; Sayada, Chalom; Philibert, Patrick; Olive, Daniel; Halfon, Philippe

doi:10.1097/qad.0000000000000267

Cited by 60 publications

(44 citation statements)

References 26 publications

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“…Conventional sequencing-based methods, including commercially available HIV-1 genotyping assays TruGene [64] and ViroSeq [65], have been widely used in the clinical practice. They detect all the mutations present in the analyzed consensus sequence of the viral population, though they are not sensitive enough to detect low-abundance mutations below a threshold of 25–30% [30,31]. Currently, NGS-based approaches are highly sensitive for detecting drug-resistance mutations in minority subpopulations [32,34,35], though these assays require highly technical/bioinformatics skills and are still too expensive to be a realistic option in most clinical laboratories.…”

Section: Discussionmentioning

confidence: 99%

“…The traditional HIV-1 population or consensus sequencing provides information limited to the genotype of the predominant or major viral variant, and it fails to detect minority subpopulations represented in less than 25% of the total quasispecies [30,31]. In turn, the sequence analysis of a representative number of molecular clones (usually, 20 to 100) derived from the amplified viral population is a very labour-intensive method, poorly adapted to high-throughput analysis in clinical laboratories.…”

Section: Introductionmentioning

confidence: 99%

“…Over the last decade, next-generation sequencing (NGS) and single-genome sequencing have revolutionized the genotypic analyses of viral quasispecies diversity, as they allow a deeper penetration (down to 0.5–1%) into the composition of the evolving mutant spectrum [32]. NGS has been successfully applied to the screening of drug-resistance mutations in HIV-1 minority genomes [31,33–35], provided that appropriate correcting algorithms have been implemented to exclude artefactual mutations introduced during the enzymatic amplification and analytical processes [36,37]. However, drawbacks of NGS-based techniques still limit their daily applicability in clinical laboratories.…”

Section: Introductionmentioning

confidence: 99%

“…Such limitations include the long time required for completing a sequencing protocol, together with the need for skilled technical personnel and expert bioinformatics support (essential for handling very large sequence datasets, analysis and interpretation). Further, the lack of standard in vitro and in silico methods, as well as the high cost of the sequencing equipment restrains the use of NGS-based techniques in clinical practice [31,38]. …”

Section: Introductionmentioning

confidence: 99%

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An Efficient Microarray-Based Genotyping Platform for the Identification of Drug-Resistance Mutations in Majority and Minority Subpopulations of HIV-1 Quasispecies

et al. 2016

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The response of human immunodeficiency virus type 1 (HIV-1) quasispecies to antiretroviral therapy is influenced by the ensemble of mutants that composes the evolving population. Low-abundance subpopulations within HIV-1 quasispecies may determine the viral response to the administered drug combinations. However, routine sequencing assays available to clinical laboratories do not recognize HIV-1 minority variants representing less than 25% of the population. Although several alternative and more sensitive genotyping techniques have been developed, including next-generation sequencing (NGS) methods, they are usually very time consuming, expensive and require highly trained personnel, thus becoming unrealistic approaches in daily clinical practice. Here we describe the development and testing of a HIV-1 genotyping DNA microarray that detects and quantifies, in majority and minority viral subpopulations, relevant mutations and amino acid insertions in 42 codons of the pol gene associated with drug- and multidrug-resistance to protease (PR) and reverse transcriptase (RT) inhibitors. A customized bioinformatics protocol has been implemented to analyze the microarray hybridization data by including a new normalization procedure and a stepwise filtering algorithm, which resulted in the highly accurate (96.33%) detection of positive/negative signals. This microarray has been tested with 57 subtype B HIV-1 clinical samples extracted from multi-treated patients, showing an overall identification of 95.53% and 89.24% of the queried PR and RT codons, respectively, and enough sensitivity to detect minority subpopulations representing as low as 5–10% of the total quasispecies. The developed genotyping platform represents an efficient diagnostic and prognostic tool useful to personalize antiviral treatments in clinical practice.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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An Efficient Microarray-Based Genotyping Platform for the Identification of Drug-Resistance Mutations in Majority and Minority Subpopulations of HIV-1 Quasispecies

et al. 2016

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“…Diagnostic sensitivity and specificity a Reagent cost for monitoring protease and reverse transcriptase with in-house methods [35,69]. b Characteristics that should be evaluated for the verification of an approved commercially available assay.…”

Section: Diagnosticsmentioning

confidence: 99%

HIV-1 genotypic drug resistance testing: digging deep, reaching wide?

Laethem

Theys

Vandamme

2015

Current Opinion in Virology

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For many years, population-based Sanger sequencing has been the golden standard for drug resistance testing within the routine follow-up of HIV-1 infected patients in resource-rich settings. Often, the data generated within this framework were subsequently used for research and surveillance purposes: to understand therapy response and to gain insights into epidemiological processes. Sanger sequencing was however ill suited for diagnostic and prognostic use in resource-limited settings (RLS) and therefore not broadly implemented. Next-generation sequencing (NGS) technologies provide high-throughput approaches by the rapid acquisition of thousands to millions of short nucleotide sequences. Depending on the experimental design, the roll-out of NGS drug resistance testing at a larger scale is feasible, providing better characterization and understanding of the evolving population of viral variants within a patient and potentially improving the prognostic value of drug resistance testing. Whether the same will become true for RLS will largely depend on affordability and sample logistics, and this may affect other mutation-specific approaches.

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Comparison of HIV-1 drug-resistance genotyping by ultra-deep sequencing and sanger sequencing using clinical samples

et al. 2017

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Sanger population sequencing (SPS) is the reference technique to monitor HIV-1-infected patients' therapy. Ultra-deep sequencing (UDS), which allows quantitative detection of drug resistance mutations, may be an alternative method. The study aimed to compare reproducibility and predictions of UDS versus SPS in a routine setting. A control containing low-abundance variants was repeatedly tested and clinical plasma samples from 100 patients were prospectively assayed by SPS and UDS using the Roche 454 system. Complete analysis by UDS was available for 88% of samples with various viral loads and subtypes. Comparison of detection thresholds found that SPS sensitivity was variable. Variations found by UDS between 5% to >20% were detected by SPS in 25% to more than 80% of samples. At the 5% cut-off, disagreements were rare and in most cases UDS detected an additional protease secondary mutation, suggesting a possible resistance to a protease inhibitor according to the 2015 ANRS algorithm. Mutations found on reverse transcriptase by only UDS were often explained by previous therapy. UDS with a variant detection threshold at 5% might allow therapy management with minimal differences compared to population sequencing while providing additional information for further determination of pertinent cutoff values for specific resistance mutations.

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Comparison of ultra-deep versus Sanger sequencing detection of minority mutations on the HIV-1 drug resistance interpretations after virological failure

Cited by 60 publications

References 26 publications

An Efficient Microarray-Based Genotyping Platform for the Identification of Drug-Resistance Mutations in Majority and Minority Subpopulations of HIV-1 Quasispecies

An Efficient Microarray-Based Genotyping Platform for the Identification of Drug-Resistance Mutations in Majority and Minority Subpopulations of HIV-1 Quasispecies

HIV-1 genotypic drug resistance testing: digging deep, reaching wide?

Comparison of HIV-1 drug-resistance genotyping by ultra-deep sequencing and sanger sequencing using clinical samples

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