Article

Wang C, Mitsuya Y, Gharizadeh B, et al.. Characterization of mutation spectra with ultra-deep pyrosequencing: application to HIV-1 drug resistance

Division of Infectious Diseases, Department of Medicine, Stanford University, Stanford, CA 94305, USA.
Genome Research (Impact Factor: 14.63). 09/2007; 17(8):1195-201. DOI: 10.1101/gr.6468307
Source: PubMed

ABSTRACT

The detection of mutant spectra within a population of microorganisms is critical for the management of drug-resistant infections. We performed ultra-deep pyrosequencing to detect minor sequence variants in HIV-1 protease and reverse transcriptase (RT) genes from clinical plasma samples. We estimated empirical error rates from four HIV-1 plasmid clones and used them to develop a statistical approach to distinguish authentic minor variants from sequencing errors in eight clinical samples. Ultra-deep pyrosequencing detected an average of 58 variants per sample compared with an average of eight variants per sample detected by conventional direct-PCR dideoxynucleotide sequencing. In the clinical sample with the largest number of minor sequence variants, all 60 variants present in > or =3% of genomes and 20 of 35 variants present in <3% of genomes were confirmed by limiting dilution sequencing. With appropriate analysis, ultra-deep pyrosequencing is a promising method for characterizing genetic diversity and detecting minor yet clinically relevant variants in biological samples with complex genetic populations.

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    • "These subpopulations in low-abundance can lead to mutations associated with antiretroviral resistance and can develop rapidly to become predominant under conditions of selective pressure which eventually lead to therapeutic failure[18]. An alternative to identify these minority variants is the use of ultra-deep sequencing, also known as next-generation sequencing (NGS) technologies[19,20,21,22]. The possibility of detecting mutations associated with resistance to antiretrovirals drugs of various viralquasispecies, including those of low-abundance is the main advantage of the ultra-deep sequencing over the traditional Sanger method. "

    Full-text · Dataset · Jan 2016
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    • "These subpopulations in low-abundance can lead to mutations associated with antiretroviral resistance and can develop rapidly to become predominant under conditions of selective pressure which eventually lead to therapeutic failure [18]. An alternative to identify these minority variants is the use of ultra-deep sequencing, also known as next-generation sequencing (NGS) technologies [19, 20, 21, 22]. The possibility of detecting mutations associated with resistance to antiretrovirals drugs of various viral quasispecies, including those of low-abundance is the main advantage of the ultra-deep sequencing over the traditional Sanger method. "
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    ABSTRACT: Although Structured Treatment Interruptions (STI) are currently not considered an alternative strategy for antiretroviral treatment, their true benefits and limitations have not been fully established. Some studies suggest the possibility of improving the quality of life of patients with this strategy; however, the information that has been obtained corresponds mostly to studies conducted in adults, with a lack of knowledge about its impact on children. Furthermore, mutations associated with antiretroviral resistance could be selected due to sub-therapeutic levels of HAART at each interruption period. Genotyping methods to determine the resistance profiles of the infecting viruses have become increasingly important for the management of patients under STI, thus low-abundance antiretroviral drug-resistant mutations (DRM's) at levels under limit of detection of conventional genotyping (<20% of quasispecies) could increase the risk of virologic failure. In this work, we analyzed the protease and reverse transcriptase regions of the pol gene by ultra-deep sequencing in pediatric patients under STI with the aim of determining the presence of high- and low-abundance DRM's in the viral rebounds generated by the STI. High-abundance mutations in protease and high- and low-abundance mutations in reverse transcriptase were detected but no one of these are directly associated with resistance to antiretroviral drugs. The results could suggest that the evaluated STI program is virologically safe, but strict and carefully planned studies, with greater numbers of patients and interruption/restart cycles, are still needed to evaluate the selection of DRM's during STI.
    Full-text · Article · Jan 2016 · PLoS ONE
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    • "The advent of so-called ''next-generation'' sequencing (NGS) technologies, with their potential to survey thousands of viral sequences from a given host, has dramatically improved our ability to characterize within-host sequence diversity in viral infections . NGS has been applied to address such questions as overall viral diversity within-hosts (Lauck et al., 2012; Wright et al., 2011); evolution of T-cell epitopes under selection by the host immune system (Bimber et al., 2010; Hughes et al., 2010, 2012; Mudd et al., 2012; O'Connor et al., 2012; Walsh et al., 2013); response of viruses to selection imposed by antiviral drugs (Cannon et al., 2008; Hedskog et al., 2010; Le et al., 2009; Wang et al., 2010a); differences between virus subpopulations infecting different host cell types (Rozera et al., 2009); and population bottlenecks in infection (Wang et al., 2010b). Here we discuss statistical methods for using NGS data to understand nucleotide sequence diversity of within-host viral populations , with particular emphasis on the comparison of synonymous and nonsynonymous (amino acid-altering) nucleotide diversity in coding regions. "
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    ABSTRACT: Next-generation sequencing (NGS) technology offers new opportunities for understanding the evolution and dynamics of viral populations within individual hosts over the course of infection. We review simple methods for estimating synonymous and nonsynonymous nucleotide diversity in viral genes from NGS data without the need for inferring linkage. We discuss the potential usefulness of these data for addressing questions of both practical and theoretical interest, including fundamental questions regarding the effective population sizes of within-host viral populations and the modes of natural selection acting on them. Copyright © 2014. Published by Elsevier B.V.
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