TY - JOUR
T1 - Next-generation sequencing of HIV-1 single genome amplicons
AU - Kijak, Gustavo H.
AU - Sanders-Buell, Eric
AU - Pham, Phuc
AU - Harbolick, Elizabeth A.
AU - Oropeza, Celina
AU - O'Sullivan, Anne Marie
AU - Bose, Meera
AU - Beckett, Charmagne G.
AU - Milazzo, Mark
AU - Robb, Merlin L.
AU - Peel, Sheila A.
AU - Scott, Paul T.
AU - Michael, Nelson L.
AU - Armstrong, Adam W.
AU - Kim, Jerome H.
AU - Brett-Major, David M.
AU - Tovanabutra, Sodsai
N1 - Publisher Copyright:
© 2019
PY - 2019/3
Y1 - 2019/3
N2 - 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.
AB - 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.
KW - Bioinformatics
KW - HIV-1
KW - IonTorrent
KW - Next-generation sequencing
KW - Single genome amplification
UR - http://www.scopus.com/inward/record.url?scp=85062687071&partnerID=8YFLogxK
U2 - 10.1016/j.bdq.2019.01.002
DO - 10.1016/j.bdq.2019.01.002
M3 - Article
AN - SCOPUS:85062687071
SN - 2214-7535
VL - 17
JO - Biomolecular Detection and Quantification
JF - Biomolecular Detection and Quantification
M1 - 100080
ER -