Next-Generation Sequencing of HIV-1 Single Genome Amplicons

Authors

Gustavo H. Kijak, Walter Reed Army Institute of Research
Eric Sanders-Buell, Walter Reed Army Institute of Research
Phuc Pham, Walter Reed Army Institute of Research
Elizabeth A. Harbolick, Walter Reed Army Institute of Research
Celina Oropeza, Walter Reed Army Institute of Research
Anne Marie O'Sullivan, Walter Reed Army Institute of Research
Meera Bose, Walter Reed Army Institute of Research
Charmagne G. Beckett, Navy Bloodborne Infection Management Center
Mark Milazzo, Walter Reed Army Institute of Research
Merlin L. Robb, Walter Reed Army Institute of Research
Sheila A. Peel, Walter Reed Army Institute of Research
Paul T. Scott, Walter Reed Army Institute of Research
Nelson L. Michael, Walter Reed Army Institute of Research
Adam W. Armstrong, Navy Medical Research Unit 6
Jerome H. Kim, Walter Reed Army Institute of Research
David Brett-Major, University of Nebraska Medical CenterFollow
Sodsai Tovanabutra, Walter Reed Army Institute of Research

Document Type

Article

Journal Title

Biomolecular Detection and Quantification

Publication Date

2019

Volume

17

Abstract

The analysis of HIV-1 sequences has helped understand the viral molecular epidemiology, monitor the development of antiretroviral drug resistance, and design candidate vaccines. The introduction of single genome amplification (SGA) has been a major advancement in the field, allowing for the characterization of multiple sequences per patient while preserving linkage among polymorphisms in the same viral genome copy. Sequencing of SGA amplicons is performed by capillary Sanger sequencing, which presents low throughput, requires a high amount of template, and is highly sensitive to template/primer mismatching. In order to meet the increasing demand for HIV-1 SGA amplicon sequencing, we have developed a platform based on benchtop next-generation sequencing (NGS) (IonTorrent) accompanied by a bioinformatics pipeline capable of running on computer resources commonly available at research laboratories. During assay validation, the NGS-based sequencing of 10 HIV-1 env SGA amplicons was fully concordant with Sanger sequencing. The field test was conducted on plasma samples from 10 US Navy and Marine service members with recent HIV-1 infection (sampling interval: 2005-2010; plasma viral load: 5,884-194,984 copies/ml). The NGS analysis of 101 SGA amplicons (median: 10 amplicons/individual) showed within-individual viral sequence profiles expected in individuals at this disease stage, including individuals with highly homogeneous quasispecies, individuals with two highly homogeneous viral lineages, and individuals with heterogeneous viral populations. In a scalability assessment using the Ion Chef automated system, 41/43 tested env SGA amplicons (95%) multiplexed on a single Ion 318 chip showed consistent gene-wide coverage >50×. With lower sample requirements and higher throughput, this approach is suitable to support the increasing demand for high-quality and cost-effective HIV-1 sequences in fields such as molecular epidemiology, and development of preventive and therapeutic strategies.

DOI Link

10.1016/j.bdq.2019.01.002

ISSN

2214-7535

Creative Commons License

This work is licensed under a Creative Commons Public Domain Dedication 1.0 License.

Rights

U.S. Government Work

Recommended Citation

Kijak, Gustavo H.; Sanders-Buell, Eric; Pham, Phuc; Harbolick, Elizabeth A.; Oropeza, Celina; O'Sullivan, Anne Marie; Bose, Meera; Beckett, Charmagne G.; Milazzo, Mark; Robb, Merlin L.; Peel, Sheila A.; Scott, Paul T.; Michael, Nelson L.; Armstrong, Adam W.; Kim, Jerome H.; Brett-Major, David; and Tovanabutra, Sodsai, "Next-Generation Sequencing of HIV-1 Single Genome Amplicons" (2019). Journal Articles: Epidemiology. 39.
https://digitalcommons.unmc.edu/coph_epidem_articles/39