Frontiers in Microbiology (Nov 2015)

Quasispecies Analyses of the HIV-1 Near-full-length Genome with Illumina MiSeq

  • Hirotaka eOde,
  • Masakazu eMatsuda,
  • Kazuhiro eMatsuoka,
  • Atsuko eHachiya,
  • Junko eHattori,
  • Yumiko eKito,
  • Yoshiyuki eYokomaku,
  • Yasumasa eIwatani,
  • Yasumasa eIwatani,
  • Wataru eSugiura,
  • Wataru eSugiura

DOI
https://doi.org/10.3389/fmicb.2015.01258
Journal volume & issue
Vol. 6

Abstract

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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 a HIV-1 clone showed that the analysis method reduced erroneous base prevalence below 1% in each sequence position and discarded only 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.

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