title: Targeted deep sequencing of HIV-1 using the IonTorrentPGM platform.
abstract: The characterization of mixed HIV-1 populations is a key question in clinical and basic research settings. This can be achieved through targeted deep sequencing (TDS), where next-generation sequencing is used to examine in depth a sub-genomic region of interest. This study explores the suitability of IonTorrent PGM(LifeTechnologies) for the TDS-based analysis of HIV-1 evolution. Using laboratory reagents and primary specimens sampled at pre-peak viremia the error rates from misincorporation and in vitro recombination were <0.5%. The sequencing error rate was 2- to 3-fold higher in/around homopolymeric tracts, and could be discerned from true polymorphism using bidirectional sequencing. The limit of detection of complex variants was further lowered by using haplotyping. The application of this system was illustrated on primary samples from an individual infected with HIV-1 followed from pre-peak viremia through six months post-acquisition. TDS provided an augmented view of the extent of genetic diversity, the covariation among polymorphisms, the evolutionary pathways, and the boundaries of the mutational space explored by the viral swarm. Based on its performance, the system can be applied for the characterization of minor viral variants in support of studies of viral evolution, which can inform the rational design of the next generation of vaccines and therapeutics.
authors.affiliation: U.S. Military HIV Research Program, Henry M. Jackson Foundation for the Advancement of Military Medicine, Bethesda, MD, USA. Electronic address: gkijak@hivresearch.org.
authors.forename: Gustavo H
authors.initials: GH
authors.lastname: Kijak
authors.affiliation: U.S. Military HIV Research Program, Henry M. Jackson Foundation for the Advancement of Military Medicine, Bethesda, MD, USA.
authors.forename: Eric
authors.initials: E
authors.lastname: Sanders-Buell
authors.affiliation: U.S. Military HIV Research Program, Henry M. Jackson Foundation for the Advancement of Military Medicine, Bethesda, MD, USA.
authors.forename: Elizabeth A
authors.initials: EA
authors.lastname: Harbolick
authors.affiliation: U.S. Military HIV Research Program, Henry M. Jackson Foundation for the Advancement of Military Medicine, Bethesda, MD, USA.
authors.forename: Phuc
authors.initials: P
authors.lastname: Pham
authors.affiliation: U.S. Military HIV Research Program, Henry M. Jackson Foundation for the Advancement of Military Medicine, Bethesda, MD, USA.
authors.forename: Agnes L
authors.initials: AL
authors.lastname: Chenine
authors.affiliation: U.S. Military HIV Research Program, Henry M. Jackson Foundation for the Advancement of Military Medicine, Bethesda, MD, USA.
authors.forename: Leigh Anne
authors.initials: LA
authors.lastname: Eller
authors.affiliation: Kenya Medical Research Institute/Walter Reed Project, Kericho, Kenya.
authors.forename: Kathleen
authors.initials: K
authors.lastname: Rono
authors.affiliation: U.S. Military HIV Research Program, Henry M. Jackson Foundation for the Advancement of Military Medicine, Bethesda, MD, USA.
authors.forename: Merlin L
authors.initials: ML
authors.lastname: Robb
authors.affiliation: U.S. Military HIV Research Program, Walter Reed Army Institute of Research, Silver Spring, MD, USA.
authors.forename: Nelson L
authors.initials: NL
authors.lastname: Michael
authors.affiliation: U.S. Military HIV Research Program, Walter Reed Army Institute of Research, Silver Spring, MD, USA.
authors.forename: Jerome H
authors.initials: JH
authors.lastname: Kim
authors.affiliation: U.S. Military HIV Research Program, Henry M. Jackson Foundation for the Advancement of Military Medicine, Bethesda, MD, USA.
authors.forename: Sodsai
authors.initials: S
authors.lastname: Tovanabutra
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D019143:Evolution, Molecular; D005260:Female; D015723:Gene Library; D014644:Genetic Variation; D015658:HIV Infections / Q000821:virology*; D015497:HIV-1 / Q000235:genetics*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D008137:Longitudinal Studies; D009154:Mutation; D011995:Recombination, Genetic; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D014766:Viremia
keywords: HIV-1; IonTorrent; Molecular evolution; Next-generation sequencing; Targeted deep sequencing