Dataset Information

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.

SUBMITTER: Ode H

PROVIDER: S-EPMC4641896 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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

Ode Hirotaka H Matsuda Masakazu M Matsuoka Kazuhiro K Hachiya Atsuko A Hattori Junko J Kito Yumiko Y Yokomaku Yoshiyuki Y Iwatani Yasumasa Y Sugiura Wataru W

Frontiers in microbiology 20151112

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 so ...[more]

PMID: 26617593

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Project description:In an effort to evaluate the accuracy of HIV-1 phylogenies based on genomes of increasing length, we developed a comprehensive near-full-length HIV-1 genome RT-PCR assay and performed a comparative evaluation via phylogenetic analyses. To this end, we conducted comparative analyses of HIV-1 phylogenies derived based on HIV-1 PR/RT (2253-3359 in the HXB2 genome) and pol region (2253-5250 in the HXB2 genome) sequences isolated from 134 HIV-1-infected patients in Cyprus (2017-2019). The HIV-1 genotypic subtypes determined using six subtyping tools (REGA 3.0, COMET 2.3, jpHMM, SCUEAL, Stanford, and Geno2pheno) were compared to investigate the discrepancies generated among different tools. To evaluate the accuracy of defined HIV-1 phylogenies, the samples exhibiting at least one discrepant subtyping result among different subtyping tools in both PR/RT and pol regions or only in the pol region (n = 38) were selected for near-full-length HIV-1 genome (790-8795 in HXB2 genome) sequencing using a newly developed RT-PCR/sequencing assay. The obtained sequences were employed for HIV-1 genotypic subtype determination and subjected to comparative phylogenetic-based analyses. It was observed that 39.6% of the 134 samples presented discrepancies in the PR/RT region, while 28.4% presented discrepancies in the pol region. REGA 3.0 produced the fewest discrepancies collectively in both regions and was selected for subsequent subtyping and comparative phylogenetic analyses of near-full-length HIV-1 genome sequences. The analyses of near-full-length HIV-1 genome sequences identified 68.4% of the 38 'discrepant samples' (n = 26) as belonging to uncharacterized recombinant HIV-1 strains, while 21.1% were circulating recombinant forms (CRFs) (n = 8) and 10.5% belonged to pure group M subtypes (n = 4). The findings demonstrated a significant reduction of 11.2% in discrepancies when pol region sequences were used compared to PR/RT region sequences, indicating that increased nucleotide sequence lengths are directly correlated with more consistent subtype classification. The results also revealed that if the discrepancy in pol region subtyping results persists, then there is a high likelihood (89.5%) that the query sequence is a recombinant HIV-1 strain, 68.4% of which belong to uncharacterized recombinant HIV-1 strains. The results of this study showed that REGA 3.0 presented the best performance in subtyping recombinant HIV-1 strains, while Stanford performed better in defining phylogenies of pure group M subtypes. The study highlights that, especially in populations with polyphyletic HIV-1 epidemics resulting in a high prevalence of recombinant HIV-1 strains, neither PR/RT nor pol region sequences are reliable for the determination of HIV-1 genotypic subtypes in samples showing discrepancies among different subtyping tools, and only near-full-length or full-length HIV-1 genome sequences are sufficiently accurate.

Dataset Information

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

Publications

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

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