scholarly journals Performance of a high-throughput next-generation sequencing method for analysis of HIV drug resistance and viral load

2020 ◽  
Vol 75 (12) ◽  
pp. 3510-3516 ◽  
Author(s):  
Jessica M Fogel ◽  
David Bonsall ◽  
Vanessa Cummings ◽  
Rory Bowden ◽  
Tanya Golubchik ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Viral Load ◽  
Next Generation Sequencing ◽  
High Throughput ◽  
Whole Genome ◽  
Hiv Viral Load ◽  
Viral Loads ◽  
Viral Load Assay ◽  
Generation Sequencing ◽  
Hiv 1

Abstract Objectives To evaluate the performance of a high-throughput research assay for HIV drug resistance testing based on whole genome next-generation sequencing (NGS) that also quantifies HIV viral load. Methods Plasma samples (n = 145) were obtained from HIV-positive MSM (HPTN 078). Samples were analysed using clinical assays (the ViroSeq HIV-1 Genotyping System and the Abbott RealTime HIV-1 Viral Load assay) and a research assay based on whole-genome NGS (veSEQ-HIV). Results HIV protease and reverse transcriptase sequences (n = 142) and integrase sequences (n = 138) were obtained using ViroSeq. Sequences from all three regions were obtained for 100 (70.4%) of the 142 samples using veSEQ-HIV; results were obtained more frequently for samples with higher viral loads (93.5% for 93 samples with >5000 copies/mL; 50.0% for 26 samples with 1000–5000 copies/mL; 0% for 23 samples with <1000 copies/mL). For samples with results from both methods, drug resistance mutations (DRMs) were detected in 33 samples using ViroSeq and 42 samples using veSEQ-HIV (detection threshold: 5.0%). Overall, 146 major DRMs were detected; 107 were detected by both methods, 37 were detected by veSEQ-HIV only (frequency range: 5.0%–30.6%) and two were detected by ViroSeq only. HIV viral loads estimated by veSEQ-HIV strongly correlated with results from the Abbott RealTime Viral Load assay (R2 = 0.85; n = 142). Conclusions The NGS-based veSEQ-HIV method provided results for most samples with higher viral loads, was accurate for detecting major DRMs, and detected mutations at lower levels compared with a method based on population sequencing. The veSEQ-HIV method also provided HIV viral load data.

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 High-Throughput Next-Generation Sequencing of Polioviruses

2016 ◽  
Vol 55 (2) ◽  
pp. 606-615 ◽  
Author(s):  
Anna M. Montmayeur ◽  
Terry Fei Fan Ng ◽  
Alexander Schmidt ◽  
Kun Zhao ◽  
Laura Magaña ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
High Throughput ◽  
Acid Extraction ◽  
Whole Genome ◽  
Next Generation ◽  
Diverse Range ◽  
Fta Cards ◽  
Reference Length ◽  
Dnase Treatment ◽  
Generation Sequencing

ABSTRACTThe poliovirus (PV) is currently targeted for worldwide eradication and containment. Sanger-based sequencing of the viral protein 1 (VP1) capsid region is currently the standard method for PV surveillance. However, the whole-genome sequence is sometimes needed for higher resolution global surveillance. In this study, we optimized whole-genome sequencing protocols for poliovirus isolates and FTA cards using next-generation sequencing (NGS), aiming for high sequence coverage, efficiency, and throughput. We found that DNase treatment of poliovirus RNA followed by random reverse transcription (RT), amplification, and the use of the Nextera XT DNA library preparation kit produced significantly better results than other preparations. The average viral reads per total reads, a measurement of efficiency, was as high as 84.2% ± 15.6%. PV genomes covering >99 to 100% of the reference length were obtained and validated with Sanger sequencing. A total of 52 PV genomes were generated, multiplexing as many as 64 samples in a single Illumina MiSeq run. This high-throughput, sequence-independent NGS approach facilitated the detection of a diverse range of PVs, especially for those in vaccine-derived polioviruses (VDPV), circulating VDPV, or immunodeficiency-related VDPV. In contrast to results from previous studies on other viruses, our results showed that filtration and nuclease treatment did not discernibly increase the sequencing efficiency of PV isolates. However, DNase treatment after nucleic acid extraction to remove host DNA significantly improved the sequencing results. This NGS method has been successfully implemented to generate PV genomes for molecular epidemiology of the most recent PV isolates. Additionally, the ability to obtain full PV genomes from FTA cards will aid in facilitating global poliovirus surveillance.

scholarly journals Correction: First evaluation of the Next-Generation Sequencing platform for the detection of HIV-1 drug resistance mutations in Belgium

PLoS ONE ◽  
2019 ◽  
Vol 14 (1) ◽  
pp. e0211587
Author(s):  
Géraldine Dessilly ◽  
Léonie Goeminne ◽  
Anne-thérèse Vandenbroucke ◽  
François E. Dufrasne ◽  
Anandi Martin ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Next Generation Sequencing ◽  
Next Generation ◽  
Resistance Mutations ◽  
Sequencing Platform ◽  
Drug Resistance Mutations ◽  
Next Generation Sequencing Platform ◽  
Generation Sequencing ◽  
Hiv 1

scholarly journals Application of a Sanger-Based External Quality Assurance Strategy for the Transition of HIV-1 Drug Resistance Assays to Next Generation Sequencing

Viruses ◽  
2020 ◽  
Vol 12 (12) ◽  
pp. 1456
Author(s):  
Cheryl Jennings ◽  
Neil T. Parkin ◽  
Daniel J. Zaccaro ◽  
Rupert Capina ◽  
Paul Sandstrom ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Quality Assurance ◽  
Next Generation Sequencing ◽  
Proficiency Testing ◽  
External Quality Assurance ◽  
External Quality ◽  
Assay Performance ◽  
Variant Detection ◽  
Generation Sequencing ◽  
Hiv 1

The National Institute of Allergy and Infectious Diseases (NIAID) Virology Quality Assurance (VQA) established a robust proficiency testing program for Sanger sequencing (SS)-based HIV-1 drug resistance (HIVDR) testing in 2001. While many of the lessons learned during the development of such programs may also apply to next generation sequencing (NGS)-based HIVDR assays, challenges remain for the ongoing evaluation of NGS-based testing. These challenges include a proper assessment of assay accuracy and the reproducibility of low abundance variant detection, intra- and inter-assay performance comparisons among laboratories using lab-defined tests, and different data analysis pipelines designed for NGS. In collaboration with the World Health Organization (WHO) Global HIVDR Laboratory Network and the Public Health Agency of Canada, the Rush VQA program distributed archived proficiency testing panels to ten laboratories to evaluate internally developed NGS assays. Consensus FASTA files were submitted using 5%, 10%, and 20% variant detection thresholds, and scored based on the same criteria used for SS. This small study showed that the SS External Quality Assurance (EQA) approach can be used as a transitional strategy for using NGS to generate SS-like data and for ongoing performance while using NGS data from the same quality control materials to further evaluate NGS assay performance.

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