title: Quasispecies Analyses of the HIV-1 Near-full-length Genome With Illumina MiSeq.
abstract: Human immunodeficiency virus type-1 (HIV-1) exhibits high between-host genetic diversity and within-host heterogeneity, recognized as quasispecies. Because HIV-1 quasispecies fluctuate in terms of multiple factors, such as antiretroviral exposure and host immunity, analyzing the HIV-1 genome is critical for selecting effective antiretroviral therapy and understanding within-host viral coevolution mechanisms. Here, to obtain HIV-1 genome sequence information that includes minority variants, we sought to develop a method for evaluating quasispecies throughout the HIV-1 near-full-length genome using the Illumina MiSeq benchtop deep sequencer. To ensure the reliability of minority mutation detection, we applied an analysis method of sequence read mapping onto a consensus sequence derived from de novo assembly followed by iterative mapping and subsequent unique error correction. Deep sequencing analyses of aHIV-1 clone showed that the analysis method reduced erroneous base prevalence below 1% in each sequence position and discarded only < 1% of all collected nucleotides, maximizing the usage of the collected genome sequences. Further, we designed primer sets to amplify the HIV-1 near-full-length genome from clinical plasma samples. Deep sequencing of 92 samples in combination with the primer sets and our analysis method provided sufficient coverage to identify >1%-frequency sequences throughout the genome. When we evaluated sequences of pol genes from 18 treatment-naïve patients' samples, the deep sequencing results were in agreement with Sanger sequencing and identified numerous additional minority mutations. The results suggest that our deep sequencing method would be suitable for identifying within-host viral population dynamics throughout the genome.
authors.affiliation: Department of Infectious Diseases and Immunology, Clinical Research Center, National Hospital Organization Nagoya Medical Center Nagoya, Japan.
authors.forename: Hirotaka
authors.initials: H
authors.lastname: Ode
authors.affiliation: Department of Infectious Diseases and Immunology, Clinical Research Center, National Hospital Organization Nagoya Medical Center Nagoya, Japan.
authors.forename: Masakazu
authors.initials: M
authors.lastname: Matsuda
authors.affiliation: Department of Infectious Diseases and Immunology, Clinical Research Center, National Hospital Organization Nagoya Medical Center Nagoya, Japan.
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Matsuoka
authors.affiliation: Department of Infectious Diseases and Immunology, Clinical Research Center, National Hospital Organization Nagoya Medical Center Nagoya, Japan.
authors.forename: Atsuko
authors.initials: A
authors.lastname: Hachiya
authors.affiliation: Department of Infectious Diseases and Immunology, Clinical Research Center, National Hospital Organization Nagoya Medical Center Nagoya, Japan.
authors.forename: Junko
authors.initials: J
authors.lastname: Hattori
authors.affiliation: Department of Infectious Diseases and Immunology, Clinical Research Center, National Hospital Organization Nagoya Medical Center Nagoya, Japan.
authors.forename: Yumiko
authors.initials: Y
authors.lastname: Kito
authors.affiliation: Department of Infectious Diseases and Immunology, Clinical Research Center, National Hospital Organization Nagoya Medical Center Nagoya, Japan.
authors.forename: Yoshiyuki
authors.initials: Y
authors.lastname: Yokomaku
authors.affiliation: Department of Infectious Diseases and Immunology, Clinical Research Center, National Hospital Organization Nagoya Medical Center Nagoya, Japan ; Department of AIDS Research, Graduate School of Medicine, Nagoya University Nagoya, Japan.
authors.forename: Yasumasa
authors.initials: Y
authors.lastname: Iwatani
authors.affiliation: Department of Infectious Diseases and Immunology, Clinical Research Center, National Hospital Organization Nagoya Medical Center Nagoya, Japan ; Department of AIDS Research, Graduate School of Medicine, Nagoya University Nagoya, Japan.
authors.forename: Wataru
authors.initials: W
authors.lastname: Sugiura
keywords: HIV-1; consensus sequence estimation; deep sequencing; drug resistance; error correction; quasispecies