RNA and DNA Sanger sequencing versus next-generation sequencing for HIV-1 drug resistance testing in treatment-naive patients

Alidjinou, Enagnon Kazali; Deldalle, Joséphine; Hallaert, Christophe; Robineau, Olivier; Ajana, F.; Choisy, P.; Hober, Didier; Bocket, Laurence

doi:10.1093/jac/dkx232

Cited by 32 publications

(24 citation statements)

References 45 publications

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“…The resulting sam files were converted to bam files, sorted and indexed using samtools, and the sorted bam files were converted to fastq files using the bamtools software [24,25]. The resulting fastq files were analyzed with Hydra Web (Government of Canada, Ottawa, Canada) with 20% and 5% sensitivity threshold for reporting low-frequency variants [26,27,28] We used the default target coverage (10,000 reads) and filtering settings (length cutoff: 100; score cutoff: 30) in HyDRA [29]. …”

Section: Methodsmentioning

confidence: 99%

Sanger and Next Generation Sequencing Approaches to Evaluate HIV-1 Virus in Blood Compartments

Arias

López

Sánchez

et al. 2018

IJERPH

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The implementation of antiretroviral treatment combined with the monitoring of drug resistance mutations improves the quality of life of HIV-1 positive patients. The drug resistance mutation patterns and viral genotypes are currently analyzed by DNA sequencing of the virus in the plasma of patients. However, the virus compartmentalizes, and different T cell subsets may harbor distinct viral subsets. In this study, we compared the patterns of HIV distribution in cell-free (blood plasma) and cell-associated viruses (peripheral blood mononuclear cells, PBMCs) derived from ART-treated patients by using Sanger sequencing- and Next-Generation sequencing-based HIV assay. CD4+CD45RA−RO+ memory T-cells were isolated from PBMCs using a BD FACSAria instrument. HIV pol (protease and reverse transcriptase) was RT-PCR or PCR amplified from the plasma and the T-cell subset, respectively. Sequences were obtained using Sanger sequencing and Next-Generation Sequencing (NGS). Sanger sequences were aligned and edited using RECall software (beta v3.03). The Stanford HIV database was used to evaluate drug resistance mutations. Illumina MiSeq platform and HyDRA Web were used to generate and analyze NGS data, respectively. Our results show a high correlation between Sanger sequencing and NGS results. However, some major and minor drug resistance mutations were only observed by NGS, albeit at different frequencies. Analysis of low-frequency drugs resistance mutations and virus distribution in the blood compartments may provide information to allow a more sustainable response to therapy and better disease management.

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Section: Methodsmentioning

confidence: 99%

Sanger and Next Generation Sequencing Approaches to Evaluate HIV-1 Virus in Blood Compartments

Arias

López

Sánchez

et al. 2018

IJERPH

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“…The average number of haplotypes per participant was the same for PR/RT and int (2 haplotypes) and slightly higher for env (4). Four of the six participants that had a higher number of haplotypes reconstructed (7)(8)(9)(10)(11)(12) haplotypes in one or more gene regions) also had a higher average haplotype diversity estimate of 0.634 (range: 0.392-0.777), while the other two had a very low average haplotype diversity estimate (0.032, range: 0.027-0.038). Participants with HRH and MSM risk factors were found to have an average of 3 reconstructed haplotypes, with haplotype diversities of 0.338 and 0.335, respectively.…”

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confidence: 97%

A cross-sectional study to characterize local HIV-1 dynamics in Washington, DC using next-generation sequencing

Gibson

Jair

Castel

et al. 2020

Sci Rep

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the DC Cohort Executive Committee † Washington, DC continues to experience a generalized HIV-1 epidemic. We characterized the local phylodynamics of HIV-1 in DC using next-generation sequencing (NGS) data. Viral samples from 68 participants from 2016 through 2017 were sequenced and paired with epidemiological data. Phylogenetic and network inferences, drug resistant mutations (DRMs), subtypes and HIV-1 diversity estimations were completed. Haplotypes were reconstructed to infer transmission clusters. Phylodynamic inferences based on the HIV-1 polymerase (pol) and envelope genes (env) were compared. Higher HIV-1 diversity (n.s.) was seen in men who have sex with men, heterosexual, and male participants in DC. 54.0% of the participants contained at least one DRM. The 40-49 year-olds showed the highest prevalence of DRMs (22.9%). Phylogenetic analysis of pol and env sequences grouped 31.9-33.8% of the participants into clusters. HIV-TRACE grouped 2.9-12.8% of participants when using consensus sequences and 9.0-64.2% when using haplotypes. NGS allowed us to characterize the local phylodynamics of HIV-1 in DC more broadly and accurately, given a better representation of its diversity and dynamics. Reconstructed haplotypes provided novel and deeper phylodynamic insights, which led to networks linking a higher number of participants. Our understanding of the HIV-1 epidemic was expanded with the powerful coupling of HIV-1 NGS data with epidemiological data.Despite recent reductions in HIV-1 prevalence in Washington, DC from 2.5% in 2013 1 to 1.8% in 2018, the United States (US) capital is still experiencing a generalized HIV-1 epidemic -as defined by the World Health Organization 2-4 . There were 340 newly diagnosed cases in DC in 2018, and the DC rate is five times higher than the national rate 3 . Blacks, men, men who have sex with men (MSM), and heterosexuals (HRH) account for the majority of people living with HIV-1 (PLWH) in DC 2,3 . However, ~20% of the newly diagnosed persons had an unknown risk for transmission in both 2016 and 2017 2,3 . Furthermore, the leading group (33.3%) of newly diagnosed cases was between the ages of 20-29 years old 3 . This same age group had the highest percentage (27.5%) of drug resistance mutations (DRM) at diagnosis, suggesting broader spread of HIV-1 drug resistant variants and potential concern for future therapeutic options, especially if these mutations are against first line antiretroviral (ART) drugs for newly infected individuals. With blacks and young adults being the most impacted groups of individuals for HIV-1 in DC, understanding the current HIV-1 phylodynamics can provide informative data to guide programs that prevent and reduce the incidence of HIV-1. Moreover, identifying potential transmission clusters amongst individuals in DC and their associated epidemiological features may help infer otherwise 'unknown' transmission modes and provide insight for more targeted prevention and intervention strategies.In 2011, the DC Cohort, a longitudinal observational NIH-funded co...

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“…The starting material, i.e., plasma viral RNA versus proviral DNA obtained from peripheral blood mononuclear cells (PBMC) or the buffy coat fraction, can strongly influence the detection of DRMs and HIVDR test results [ 16 ]. Population sequencing on proviral DNA can introduce bias due to the inclusion of defective virus sequences, leading to an increased proportion of hypermutation and stop codons [ 17 , 18 ], especially in samples where the contribution of proviral DNA outweighs that of plasma virus RNA.…”

Section: Laboratory Considerationsmentioning

confidence: 99%

“…However, the main drawback of this technology is its inability to reliably detect low-abundance DRMs below the 20% threshold [ 1 , 39 , 40 , 41 ]. Several studies have shown that NGS-based HIVDR testing is highly concordant to Sanger sequencing at a 20% detection threshold [ 5 , 15 , 16 , 42 , 43 ]. Thus, leveraging on the potentially improved efficiency, increased scalability, decreased cost, and higher sensitivity, many laboratories are transitioning to NGS for HIVDR testing.…”

Section: Use Of Ngs For Clinical Hivdr Testingmentioning

confidence: 99%

Next-Generation Sequencing for HIV Drug Resistance Testing: Laboratory, Clinical, and Implementation Considerations

Ávila‐Ríos¹,

Parkin

Swanstrom

et al. 2020

Viruses

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Higher accessibility and decreasing costs of next generation sequencing (NGS), availability of commercial kits, and development of dedicated analysis pipelines, have allowed an increasing number of laboratories to adopt this technology for HIV drug resistance (HIVDR) genotyping. Conventional HIVDR genotyping is traditionally carried out using population-based Sanger sequencing, which has a limited capacity for reliable detection of variants present at intra-host frequencies below a threshold of approximately 20%. NGS has the potential to improve sensitivity and quantitatively identify low-abundance variants, improving efficiency and lowering costs. However, some challenges exist for the standardization and quality assurance of NGS-based HIVDR genotyping. In this paper, we highlight considerations of these challenges as related to laboratory, clinical, and implementation of NGS for HIV drug resistance testing. Several sources of variation and bias occur in each step of the general NGS workflow, i.e., starting material, sample type, PCR amplification, library preparation method, instrument and sequencing chemistry-inherent errors, and data analysis options and limitations. Additionally, adoption of NGS-based HIVDR genotyping, especially for clinical care, poses pressing challenges, especially for resource-poor settings, including infrastructure and equipment requirements and cost, logistic and supply chains, instrument service availability, personnel training, validated laboratory protocols, and standardized analysis outputs. The establishment of external quality assessment programs may help to address some of these challenges and is needed to proceed with NGS-based HIVDR genotyping adoption.

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RNA and DNA Sanger sequencing versus next-generation sequencing for HIV-1 drug resistance testing in treatment-naive patients

Abstract: DSS does not clearly improve the detection of RAMs in ART-naive patients, as compared with RSS. NGS allows detection of additional minority RAMs; however, their clinical relevance requires further investigation.

Cited by 32 publications

References 45 publications

Sanger and Next Generation Sequencing Approaches to Evaluate HIV-1 Virus in Blood Compartments

Sanger and Next Generation Sequencing Approaches to Evaluate HIV-1 Virus in Blood Compartments

A cross-sectional study to characterize local HIV-1 dynamics in Washington, DC using next-generation sequencing

Next-Generation Sequencing for HIV Drug Resistance Testing: Laboratory, Clinical, and Implementation Considerations

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