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

2018 ◽  
Vol 56 (6) ◽  
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.

scholarly journals HIV-1 genotypic resistance testing using the Vela automated next-generation sequencing platform

2018 ◽  
Vol 73 (5) ◽  
pp. 1152-1157 ◽  
Author(s):  
Stéphanie Raymond ◽  
Florence Nicot ◽  
Romain Carcenac ◽  
Caroline Lefebvre ◽  
Nicolas Jeanne ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Resistance Testing ◽  
Next Generation ◽  
Genotypic Resistance ◽  
Sequencing Platform ◽  
Next Generation Sequencing Platform ◽  
Genotypic Resistance Testing ◽  
Generation Sequencing ◽  
Hiv 1

Routine drug resistance testing in HIV-1 proviral DNA, using an automated next- generation sequencing assay

2019 ◽  
Vol 121 ◽  
pp. 104207 ◽  
Author(s):  
Enagnon Kazali Alidjinou ◽  
Pauline Coulon ◽  
Christophe Hallaert ◽  
Olivier Robineau ◽  
Agnès Meybeck ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Next Generation Sequencing ◽  
Resistance Testing ◽  
Next Generation ◽  
Proviral Dna ◽  
Drug Resistance Testing ◽  
Generation Sequencing ◽  
Hiv 1

scholarly journals HIV-1 Protease, Reverse Transcriptase, and Integrase Variation

2016 ◽  
Vol 90 (13) ◽  
pp. 6058-6070 ◽  
Author(s):  
Soo-Yon Rhee ◽  
Kris Sankaran ◽  
Vici Varghese ◽  
Mark A. Winters ◽  
Christopher B. Hurt ◽  
...  
Keyword(s):  
Quality Control ◽  
Amino Acid ◽  
Amino Acid Position ◽  
Antiretroviral Drugs ◽  
Resistance Testing ◽  
Genotypic Resistance ◽  
Naturally Occurring ◽  
Genotypic Resistance Testing ◽  
Hiv 1 ◽  
Amino Acid Variants

ABSTRACTHIV-1 protease (PR), reverse transcriptase (RT), and integrase (IN) variability presents a challenge to laboratories performing genotypic resistance testing. This challenge will grow with increased sequencing of samples enriched for proviral DNA such as dried blood spots and increased use of next-generation sequencing (NGS) to detect low-abundance HIV-1 variants. We analyzed PR and RT sequences from >100,000 individuals and IN sequences from >10,000 individuals to characterize variation at each amino acid position, identify mutations indicating APOBEC-mediated G-to-A editing, and identify mutations resulting from selective drug pressure. Forty-seven percent of PR, 37% of RT, and 34% of IN positions had one or more amino acid variants with a prevalence of ≥1%. Seventy percent of PR, 60% of RT, and 60% of IN positions had one or more variants with a prevalence of ≥0.1%. Overall 201 PR, 636 RT, and 346 IN variants had a prevalence of ≥0.1%. The median intersubtype prevalence ratios were 2.9-, 2.1-, and 1.9-fold for these PR, RT, and IN variants, respectively. Only 5.0% of PR, 3.7% of RT, and 2.0% of IN variants had a median intersubtype prevalence ratio of ≥10-fold. Variants at lower prevalences were more likely to differ biochemically and to be part of an electrophoretic mixture compared to high-prevalence variants. There were 209 mutations indicative of APOBEC-mediated G-to-A editing and 326 mutations nonpolymorphic treatment selected. Identification of viruses with a high number of APOBEC-associated mutations will facilitate the quality control of dried blood spot sequencing. Identifying sequences with a high proportion of rare mutations will facilitate the quality control of NGS.IMPORTANCEMost antiretroviral drugs target three HIV-1 proteins: PR, RT, and IN. These proteins are highly variable: many different amino acids can be present at the same position in viruses from different individuals. Some of the amino acid variants cause drug resistance and occur mainly in individuals receiving antiretroviral drugs. Some variants result from a human cellular defense mechanism called APOBEC-mediated hypermutation. Many variants result from naturally occurring mutation. Some variants may represent technical artifacts. We studied PR and RT sequences from >100,000 individuals and IN sequences from >10,000 individuals to quantify variation at each amino acid position in these three HIV-1 proteins. We performed analyses to determine which amino acid variants resulted from antiretroviral drug selection pressure, APOBEC-mediated editing, and naturally occurring variation. Our results provide information essential to clinical, research, and public health laboratories performing genotypic resistance testing by sequencing HIV-1 PR, RT, and IN.

scholarly journals Next Generation Sequencing for HIV-1 Drug Resistance Testing—A Special Issue Walkthrough

Viruses ◽  
2021 ◽  
Vol 13 (2) ◽  
pp. 340
Author(s):  
Rami Kantor
Keyword(s):  
Drug Resistance ◽  
Next Generation Sequencing ◽  
Resistance Testing ◽  
Next Generation ◽  
Special Issue ◽  
Global Challenge ◽  
Drug Resistance Testing ◽  
Generation Sequencing ◽  
Hiv 1

Drug resistance remains a global challenge in the fight against the HIV pandemic [...]

scholarly journals External Quality Assessment for Next-Generation Sequencing-Based HIV Drug Resistance Testing: Unique Requirements and Challenges

Viruses ◽  
2020 ◽  
Vol 12 (5) ◽  
pp. 550 ◽  
Author(s):  
Emma R. Lee ◽  
Feng Gao ◽  
Paul Sandstrom ◽  
Hezhao Ji
Keyword(s):  
Drug Resistance ◽  
Next Generation Sequencing ◽  
Quality Assessment ◽  
Sanger Sequencing ◽  
External Quality Assessment ◽  
Resistance Testing ◽  
Next Generation ◽  
External Quality ◽  
Hiv Drug Resistance ◽  
Generation Sequencing

Over the past decade, there has been an increase in the adoption of next generation sequencing (NGS) technologies for HIV drug resistance (HIVDR) testing. NGS far outweighs conventional Sanger sequencing as it has much higher throughput, lower cost when samples are batched and, most importantly, significantly higher sensitivities for variants present at low frequencies, which may have significant clinical implications. Despite the advantages of NGS, Sanger sequencing remains the gold standard for HIVDR testing, largely due to the lack of standardization of NGS-based HIVDR testing. One important aspect of standardization includes external quality assessment (EQA) strategies and programs. Current EQA for Sanger-based HIVDR testing includes proficiency testing where samples are sent to labs and the performance of the lab conducting such assays is evaluated. The current methods for Sanger-based EQA may not apply to NGS-based tests because of the fundamental differences in their technologies and outputs. Sanger-based genotyping reports drug resistance mutations (DRMs) data as dichotomous, whereas NGS-based HIVDR genotyping also reports DRMs as numerical data (percent abundance). Here we present an overview of the need to develop EQA for NGS-based HIVDR testing and some unique challenges that may be encountered.

scholarly journals Implementation of Next-Generation Sequencing for Hepatitis B Virus Resistance Testing and Genotyping in a Clinical Microbiology Laboratory

2015 ◽  
Vol 54 (1) ◽  
pp. 127-133 ◽  
Author(s):  
Christopher F. Lowe ◽  
Linda Merrick ◽  
P. Richard Harrigan ◽  
Tony Mazzulli ◽  
Christopher H. Sherlock ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Hepatitis B ◽  
Sanger Sequencing ◽  
Clinical Microbiology ◽  
Resistance Testing ◽  
Clinical Microbiology Laboratory ◽  
Next Generation ◽  
Low Sensitivity ◽  
Pcr Targeting ◽  
Generation Sequencing

Sanger sequencing or DNA hybridization have been the primary modalities for hepatitis B (HBV) resistance testing and genotyping; however, there are limitations, such as low sensitivity and the inability to detect novel mutations. Next-generation sequencing (NGS) for HBV can overcome these limitations, but there is limited guidance for clinical microbiology laboratories to validate this novel technology. In this study, we describe an approach to implementing deep pyrosequencing for HBV resistance testing and genotyping in a clinical virology laboratory. A nested PCR targeting thepolregion of HBV (codons 143 to 281) was developed, and the PCR product was sequenced by the 454 Junior (Roche). Interpretation was performed by ABL TherapyEdge based on European Association for the Study of the Liver (EASL) guidelines. Previously characterized HBV samples by INNO-LiPA (LiPA) were compared to NGS with discordant results arbitrated by Sanger sequencing. Genotyping of 105 distinct samples revealed a concordance of 95.2% (100/105), with Sanger sequencing confirming the NGS result. Resistance testing by NGS was concordant with LiPA in 85% (68/80) of previously characterized samples. Additional mutations were found in 8 samples, which related to the identification of low-level mutant subpopulations present at <10% (6/8). To balance the costs of testing for the validation study, reproducibility of the NGS was investigated through an analysis of sequence variants at loci not associated with resistance in a single patient sample. Our validation approach attempts to balance costs with efficient data acquisition.

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