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Open AccessArticle

Multi-Laboratory Comparison of Next-Generation to Sanger-Based Sequencing for HIV-1 Drug Resistance Genotyping

Data First Consulting, Inc., Sebastopol, CA 95472, USA
Centro de Investigación en Enfermedades Infecciosas, Instituto Nacional de Enfermedades Respiratorias, Mexico City 14080, Mexico
National Infection Service, Public Health England, London NW9 5EQ, UK
British Columbia Centre for Excellence in HIV/AIDS, Vancouver, BC V6Z 1Y6, Canada
Division of Infectious Diseases, Faculty of Medicine, University of British Columbia, Vancouver, BC V5Z 1M9, Canada
Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
Division of AIDS, Department of Medicine, University of British Columbia, Vancouver, BC V5Z 1M9, Canada
Research Improving People’s Lives, Providence, RI 02909, USA
National Institute for Communicable Diseases, Johannesburg 2192, South Africa
National HIV and Retrovirology Laboratories at JC Wilt Infectious Diseases Research Center, Public Health Agency of Canada, Winnipeg, Manitoba R3E 3R2, Canada
Division of Infectious Diseases, Brown University Alpert Medical School, Providence, RI 02912, USA
IrsiCaixa AIDS Research Institute, Badalona, 08916 Catalonia, Spain
Center for Research Resources-Immunology Reference Laboratory, Ponce Health Sciences University-Ponce Research Institute, Ponce, PR 00716, USA
Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC 27514, USA
RTI International, Research Triangle Park, NC 27709, USA
Rush Medical College, Chicago, IL 60612, USA
Author to whom correspondence should be addressed.
Viruses 2020, 12(7), 694;
Received: 2 June 2020 / Revised: 20 June 2020 / Accepted: 24 June 2020 / Published: 27 June 2020
(This article belongs to the Special Issue Next Generation Sequencing for HIV Drug Resistance Testing)
Next-generation sequencing (NGS) is increasingly used for HIV-1 drug resistance genotyping. NGS methods have the potential for a more sensitive detection of low-abundance variants (LAV) compared to standard Sanger sequencing (SS) methods. A standardized threshold for reporting LAV that generates data comparable to those derived from SS is needed to allow for the comparability of data from laboratories using NGS and SS. Ten HIV-1 specimens were tested in ten laboratories using Illumina MiSeq-based methods. The consensus sequences for each specimen using LAV thresholds of 5%, 10%, 15%, and 20% were compared to each other and to the consensus of the SS sequences (protease 4–99; reverse transcriptase 38–247). The concordance among laboratories’ sequences at different thresholds was evaluated by pairwise sequence comparisons. NGS sequences generated using the 20% threshold were the most similar to the SS consensus (average 99.6% identity, range 96.1–100%), compared to 15% (99.4%, 88.5–100%), 10% (99.2%, 87.4–100%), or 5% (98.5%, 86.4–100%). The average sequence identity between laboratories using thresholds of 20%, 15%, 10%, and 5% was 99.1%, 98.7%, 98.3%, and 97.3%, respectively. Using the 20% threshold, we observed an excellent agreement between NGS and SS, but significant differences at lower thresholds. Understanding how variation in NGS methods influences sequence quality is essential for NGS-based HIV-1 drug resistance genotyping. View Full-Text
Keywords: HIV-1; drug resistance; genotyping; NGS HIV-1; drug resistance; genotyping; NGS
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Parkin, N.T.; Avila-Rios, S.; Bibby, D.F.; Brumme, C.J.; Eshleman, S.H.; Harrigan, P.R.; Howison, M.; Hunt, G.; Ji, H.; Kantor, R.; Ledwaba, J.; Lee, E.R.; Matías-Florentino, M.; Mbisa, J.L.; Noguera-Julian, M.; Paredes, R.; Rivera-Amill, V.; Swanstrom, R.; Zaccaro, D.J.; Zhang, Y.; Zhou, S.; Jennings, C. Multi-Laboratory Comparison of Next-Generation to Sanger-Based Sequencing for HIV-1 Drug Resistance Genotyping. Viruses 2020, 12, 694.

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