Combination of quadruplex qPCR and next-generation sequencing for qualitative and quantitative analysis of the HIV-1 latent reservoir

HIV-1 infection requires lifelong therapy with antiretroviral drugs due to the existence of a latent reservoir of transcriptionally inactive integrated proviruses. The goal of HIV-1 cure research is to eliminate or functionally silence this reservoir. To this end, there are numerous ongoing studies to evaluate immunological approaches, including monoclonal antibody therapies. Evaluating the results of these studies requires sensitive and specific measures of the reservoir. Here, we describe a relatively high-throughput combined quantitative PCR (qPCR) and next-generation sequencing method. Four different qPCR probes covering the packaging signal (PS), group-specific antigen (gag), polymerase (pol), and envelope (env) are combined in a single multiplex reaction to detect the HIV-1 genome in limiting dilution samples followed by sequence verification of individual reactions that are positive for combinations of any two of the four probes (Q4PCR). This sensitive and specific approach allows for an unbiased characterization of the HIV-1 latent reservoir.

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Quadruplex qPCR for qualitative and quantitative analysis of the HIV-1 latent reservoir

10.1101/641951 ◽

2019 ◽

Author(s):

Christian Gaebler ◽

Julio C. C. Lorenzi ◽

Thiago Y. Oliveira ◽

Lilian Nogueira ◽

Victor Ramos ◽

...

Keyword(s):

Next Generation Sequencing ◽

Quantitative Polymerase Chain Reaction ◽

Specific Antigen ◽

Latent Reservoir ◽

Next Generation ◽

Sequencing Method ◽

Generation Sequencing ◽

Hiv 1 ◽

Next Generation Sequencing Method

AbstractHIV-1 infection requires life-long therapy with anti-retroviral drugs due to the existence of a latent reservoir of transcriptionally inactive integrated proviruses. The goal of HIV-1 cure research is to eliminate or functionally silence this reservoir. To this end there are numerous ongoing studies to evaluate immunologic approaches including monoclonal antibody therapies. Evaluating the results of these studies requires sensitive and specific measures of the reservoir. Here we describe a relatively high throughput combined quantitative polymerase chain reaction (qPCR) and next generation sequencing method. Four different qPCR probes covering the packaging signal (PS), group-specific antigen (gag), polymerase (pol), and envelope (env) are combined in a single multiplex reaction to detect the HIV-1 genome in limiting dilution samples followed by sequence verification of individual reactions that are positive for combinations of any 2 of the 4 probes (Q4PCR). This sensitive and specific approach allows for an unbiased characterization of the HIV-1 latent reservoir.SummaryHIV-1 cure research seeks to decrease or eliminate the latent reservoir. The evaluation of such curative strategies requires accurate measures of the reservoir. Gaebler et al. describe a combined multicolor qPCR and next generation sequencing method that enables the sensitive and specific characterization of the HIV-1 latent reservoir.

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Combination of quadruplex qPCR and next-generation sequencing for qualitative and quantitative analysis of the HIV-1 latent reservoir

Journal of Virus Eradication ◽

10.1016/s2055-6640(20)30077-7 ◽

2019 ◽

Vol 5 ◽

pp. 3

Author(s):

C. Gaebler

Keyword(s):

Quantitative Analysis ◽

Next Generation Sequencing ◽

Latent Reservoir ◽

Qualitative And Quantitative Analysis ◽

Next Generation ◽

Qualitative And Quantitative ◽

Generation Sequencing ◽

Hiv 1

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Comparison of anIn VitroDiagnostic Next-Generation Sequencing Assay with Sanger Sequencing for HIV-1 Genotypic Resistance Testing

Journal of Clinical Microbiology ◽

10.1128/jcm.00105-18 ◽

2018 ◽

Vol 56 (6) ◽

Cited By ~ 28

Author(s):

Philip L. Tzou ◽

Pramila Ariyaratne ◽

Vici Varghese ◽

Charlie Lee ◽

Elian Rakhmanaliev ◽

...

Keyword(s):

Next Generation Sequencing ◽

Sanger Sequencing ◽

Low Frequency ◽

Antiretroviral Drugs ◽

Resistance Testing ◽

Next Generation ◽

Genotypic Resistance ◽

Genotypic Resistance Testing ◽

Generation Sequencing ◽

Hiv 1

ABSTRACTThe ability of next-generation sequencing (NGS) technologies to detect low frequency HIV-1 drug resistance mutations (DRMs) not detected by dideoxynucleotide Sanger sequencing has potential advantages for improved patient outcomes. We compared the performance of anin vitrodiagnostic (IVD) NGS assay, the Sentosa SQ HIV genotyping assay for HIV-1 genotypic resistance testing, with Sanger sequencing on 138 protease/reverse transcriptase (RT) and 39 integrase sequences. The NGS assay used a 5% threshold for reporting low-frequency variants. The level of complete plus partial nucleotide sequence concordance between Sanger sequencing and NGS was 99.9%. Among the 138 protease/RT sequences, a mean of 6.4 DRMs was identified by both Sanger and NGS, a mean of 0.5 DRM was detected by NGS alone, and a mean of 0.1 DRM was detected by Sanger sequencing alone. Among the 39 integrase sequences, a mean of 1.6 DRMs was detected by both Sanger sequencing and NGS and a mean of 0.15 DRM was detected by NGS alone. Compared with Sanger sequencing, NGS estimated higher levels of resistance to one or more antiretroviral drugs for 18.2% of protease/RT sequences and 5.1% of integrase sequences. There was little evidence for technical artifacts in the NGS sequences, but the G-to-A hypermutation was detected in three samples. In conclusion, the IVD NGS assay evaluated in this study was highly concordant with Sanger sequencing. At the 5% threshold for reporting minority variants, NGS appeared to attain a modestly increased sensitivity for detecting low-frequency DRMs without compromising sequence accuracy.

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