title: Comparison of Major and Minor Viral SNPs Identified through Single Template Sequencing and Pyrosequencing in Acute HIV-1 Infection.
abstract: Massively parallel sequencing (MPS) technologies, such as 454-pyrosequencing, allow for the identification of variants in sequence populations at lower levels than consensus sequencing and most single-template Sanger sequencing experiments. We sought to determine if the greater depth of population sampling attainable using MPS technology would allow detection of minor variants in HIV founder virus populations very early in infection in instances where Sanger sequencing detects only a single variant. We compared single nucleotide polymorphisms (SNPs) during acute HIV-1 infection from 32 subjects using both single template Sanger and 454-pyrosequencing. Pyrosequences from a median of 2400 viral templates per subject and encompassing 40% of the HIV-1 genome, were compared to a median of five individually amplified near full-length viral genomes sequenced using Sanger technology. There was no difference in the consensus nucleotide sequences over the 3.6kb compared in 84% of the subjects infected with single founders and 33% of subjects infected with multiple founder variants: among the subjects with disagreements, mismatches were found in less than 1% of the sites evaluated (of a total of nearly 117,000 sites across all subjects). The majority of the SNPs observed only in pyrosequences were present at less than 2% of the subject's viral sequence population. These results demonstrate the utility of the Sanger approach for study of early HIV infection and provide guidance regarding the design, utility and limitations of population sequencing from variable template sources, and emphasize parameters for improving the interpretation of massively parallel sequencing data to address important questions regarding target sequence evolution.
authors: Department of Microbiology, University of Washington, Seattle, WA, 98195, United States of America.
authors: Shyamala
authors: S
authors: Iyer
authors: Department of Microbiology, University of Washington, Seattle, WA, 98195, United States of America.
authors: Eleanor
authors: E
authors: Casey
authors: Department of Microbiology, University of Washington, Seattle, WA, 98195, United States of America.
authors: Heather
authors: H
authors: Bouzek
authors: Department of Microbiology, University of Washington, Seattle, WA, 98195, United States of America.
authors: Moon
authors: M
authors: Kim
authors: Department of Microbiology, University of Washington, Seattle, WA, 98195, United States of America.
authors: Wenjie
authors: W
authors: Deng
authors: Department of Microbiology, University of Washington, Seattle, WA, 98195, United States of America.
authors: Brendan B
authors: BB
authors: Larsen
authors: Department of Microbiology, University of Washington, Seattle, WA, 98195, United States of America.
authors: Hong
authors: H
authors: Zhao
authors: Department of Microbiology, University of Washington, Seattle, WA, 98195, United States of America.
authors: Roger E
authors: RE
authors: Bumgarner
authors: US Military HIV Research Program, WRAIR, Silver Spring, MD, 20910, United States of America; Henry Jackson Foundation for the Advancement of Military Medicine, Inc., Bethesda, MD, 20817, United States of America.
authors: Morgane
authors: M
authors: Rolland
authors: Department of Microbiology, University of Washington, Seattle, WA, 98195, United States of America; Department of Medicine, University of Washington, Seattle, WA, 98195, United States of America; Department of Laboratory Medicine, Seattle, WA, 98195, United States of America.
authors: James I
authors: JI
authors: Mullins
mesh_terms: D004279:DNA, Viral / Q000737:chemistry* / Q000235:genetics; D015658:HIV Infections / Q000821:virology*; D015497:HIV-1 / Q000235:genetics* / Q000302:isolation & purification; D006801:Humans; D020641:Polymorphism, Single Nucleotide; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000379:methods*
chemical_list: D004279:DNA, Viral