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Quinones-Mateu M.E.,University Hospital Translational Laboratory | Quinones-Mateu M.E.,Case Western Reserve University | Avila S.,Instituto Nacional Of Enfermedades Respiratorias | Reyes-Teran G.,Instituto Nacional Of Enfermedades Respiratorias | Martinez M.A.,Autonomous University of Barcelona
Journal of Clinical Virology | Year: 2014

Population (Sanger) sequencing has been the standard method in basic and clinical DNA sequencing for almost 40 years; however, next-generation (deep) sequencing methodologies are now revolutionizing the field of genomics, and clinical virology is no exception. Deep sequencing is highly efficient, producing an enormous amount of information at low cost in a relatively short period of time. High-throughput sequencing techniques have enabled significant contributions to multiples areas in virology, including virus discovery and metagenomics (viromes), molecular epidemiology, pathogenesis, and studies of how viruses to escape the host immune system and antiviral pressures. In addition, new and more affordable deep sequencing-based assays are now being implemented in clinical laboratories. Here, we review the use of the current deep sequencing platforms in virology, focusing on three of the most studied viruses: human immunodeficiency virus (HIV), hepatitis C virus (HCV), and influenza virus. © 2014 Elsevier B.V.


Kyeyune F.,Clinical Research Laboratories | Gibson R.M.,University of Western Ontario | Nanky I.,Clinical Research Laboratories | Venner C.,University of Western Ontario | And 17 more authors.
Antimicrobial Agents and Chemotherapy | Year: 2016

Most patients failing antiretroviral treatment in Uganda continue to fail their treatment regimen even if a dominant drug-resistant HIV-1 genotype is not detected. In a recent retrospective study, we observed that approximately 30% of HIV-infected individuals in the Joint Clinical Research Centre (Kampala, Uganda) experienced virologic failure with a susceptible HIV-1 genotype based on standard Sanger sequencing. Selection of minority drug-resistant HIV-1 variants (not detectable by Sanger sequencing) under antiretroviral therapy pressure can lead to a shift in the viral quasispecies distribution, becoming dominant members of the virus population and eventually causing treatment failure. Here, we used a novel HIV-1 genotyping assay based on deep sequencing (DeepGen) to quantify low-level drug-resistant HIV-1 variants in 33 patients failing a first-line antiretroviral treatment regimen in the absence of drug-resistant mutations, as screened by standard population-based Sanger sequencing. Using this sensitive assay, we observed that 64% (21/33) of these individuals had low-frequency (or minority) drug-resistant variants in the intrapatient HIV-1 population, which correlated with treatment failure. Moreover, the presence of these minority HIV-1 variants was associated with higher intrapatient HIV-1 diversity, suggesting a dynamic selection or fading of drug-resistant HIV-1 variants from the viral quasispecies in the presence or absence of drug pressure, respectively. This study identified lowfrequency HIV drug resistance mutations by deep sequencing in Ugandan patients failing antiretroviral treatment but lacking dominant drug resistance mutations as determined by Sanger sequencing methods. We showed that these low-abundance drugresistant viruses could have significant consequences for clinical outcomes, especially if treatment is not modified based on a susceptible HIV-1 genotype by Sanger sequencing. Therefore, we propose to make clinical decisions using more sensitive methods to detect minority HIV-1 variants. © 2016, American Society for Microbiology. All Rights Reserved.


Weber J.,Czech Institute of Organic Chemistry And Biochemistry | Rose J.D.,Case Western Reserve University | Vazquez A.C.,Kent State University | Winner D.,University Hospital Translational Laboratory | And 5 more authors.
PLoS ONE | Year: 2013

Most studies describing phenotypic resistance to integrase strand transfer inhibitors have analyzed viruses carrying only patient-derived HIV-1 integrase genes (INT-recombinant viruses). However, to date, many of the patients on INSTI-based treatment regimes, such as raltegravir (RAL), elvitegravir (EVG), and dolutegravir (DTG) are infected with multidrug-resistant HIV-1 strains. Here we analyzed the effect of drug resistance mutations in Gag (p2/NCp7/p1/p6), protease (PR), reverse transcriptase (RT), and integrase (IN) coding regions on susceptibility to INSTIs and viral replicative fitness using a novel HIV-1 phenotyping assay. Initial characterization based on site-directed mutant INSTI-resistant viruses confirmed the effect of a series of INSTI mutations on reduced susceptibility to EVG and RAL and viral replicative fitness (0.6% to 99% relative to the HIV-1NL4-3 control). Two sets of recombinant viruses containing a 3,428-bp gag-p2/NCp7/p1/p6/pol-PR/RT/IN (p2-INT) or a 1,088 bp integrase (INT) patient-derived fragment were constructed from plasma samples obtained from 27 virologic failure patients participating in a 48-week dose-ranging study of elvitegravir, GS-US-183-0105. A strong correlation was observed when susceptibility to EVG and RAL was assayed using p2-INT- vs. INT-recombinant viruses (Pearson coefficient correlation 0.869 and 0.918, P<0.0001 for EVG and RAL, respectively), demonstrating that mutations in the protease and RT have limited effect on susceptibility to these INSTIs. On the other hand, the replicative fitness of viruses harboring drug resistance mutations in PR, RT, and IN was generally impaired compared to viruses carrying only INSTI-resistance mutations. Thus, in the absence of drug pressure, drug resistance mutations in the PR and RT contribute to decrease the replicative fitness of the virus already impaired by mutations in the integrase. The use of recombinant viruses containing most or all HIV-1 regions targeted by antiretroviral drugs might be essential to understand the collective effect of epistatic interactions in multidrug-resistant viruses. © 2013 Weber et al.


Gibson R.M.,University Hospital Translational Laboratory | Weber J.,Czech Institute of Organic Chemistry And Biochemistry | Winner D.,University Hospital Translational Laboratory | Miller M.D.,Gilead Sciences Inc. | And 2 more authors.
PLoS ONE | Year: 2014

The role of HIV-1 minority variants on transmission, pathogenesis, and virologic failure to antiretroviral regimens has been explored; however, most studies of low-level HIV-1 drug-resistant variants have focused in single target regions. Here we used a novel HIV-1 genotypic assay based on deep sequencing, DEEPGEN (Gibson et al 2014 Antimicrob Agents Chemother 58:2167) to simultaneously analyze the presence of minority variants carrying mutations associated with reduced susceptibility to protease (PR), reverse transcriptase (RT), and integrase strand transfer integrase inhibitors (INSTIs), as well as HIV-1 coreceptor tropism. gag-p2/NCp7/p1/p6/ pol-PR/RT/INT and env/C2V3 PCR products were obtained from twelve heavily treatment-experienced patients experiencing virologic failure while participating in a 48-week dose-ranging study of elvitegravir (GS-US-183-0105). Deep sequencing results were compared with (i) virological response to treatment, (ii) genotyping based on population sequencing, (iii) phenotyping data using PhenoSense and VIRALARTS, and (iv) HIV-1 coreceptor tropism based on the phenotypic test VERITROP. Most patients failed the antiretroviral regimen with numerous pre-existing mutations in the PR and RT, and additionally newly acquired INSTI-resistance mutations as determined by population sequencing (mean 9.4, 5.3, and 1.4 PI- RTI-, and INSTI-resistance mutations, respectively). Interestingly, since DEEPGEN allows the accurate detection of amino acid substitutions at frequencies as low as 1% of the population, a series of additional drug resistance mutations were detected by deep sequencing (mean 2.5, 1.5, and 0.9, respectively). The presence of these low-abundance HIV-1 variants was associated with drug susceptibility, replicative fitness, and coreceptor tropism determined using sensitive phenotypic assays, enhancing the overall burden of resistance to all four antiretroviral drug classes. Further longitudinal studies based on deep sequencing tests will help to clarify (i) the potential impact of minority HIV-1 drug resistant variants in response to antiretroviral therapy and (ii) the importance of the detection of HIV minority variants in the clinical practice. © 2014 Gibson et al.


Archer J.,University of Manchester | Weber J.,Czech Institute of Organic Chemistry And Biochemistry | Henry K.,Case Western Reserve University | Winner D.,Case Western Reserve University | And 8 more authors.
PLoS ONE | Year: 2012

HIV-1 coreceptor tropism assays are required to rule out the presence of CXCR4-tropic (non-R5) viruses prior treatment with CCR5 antagonists. Phenotypic (e.g., Trofile™, Monogram Biosciences) and genotypic (e.g., population sequencing linked to bioinformatic algorithms) assays are the most widely used. Although several next-generation sequencing (NGS) platforms are available, to date all published deep sequencing HIV-1 tropism studies have used the 454™ Life Sciences/Roche platform. In this study, HIV-1 co-receptor usage was predicted for twelve patients scheduled to start a maraviroc-based antiretroviral regimen. The V3 region of the HIV-1 env gene was sequenced using four NGS platforms: 454™, PacBio® RS (Pacific Biosciences), Illumina®, and Ion Torrent™ (Life Technologies). Cross-platform variation was evaluated, including number of reads, read length and error rates. HIV-1 tropism was inferred using Geno2Pheno, Web PSSM, and the 11/24/25 rule and compared with Trofile™ and virologic response to antiretroviral therapy. Error rates related to insertions/deletions (indels) and nucleotide substitutions introduced by the four NGS platforms were low compared to the actual HIV-1 sequence variation. Each platform detected all major virus variants within the HIV-1 population with similar frequencies. Identification of non-R5 viruses was comparable among the four platforms, with minor differences attributable to the algorithms used to infer HIV-1 tropism. All NGS platforms showed similar concordance with virologic response to the maraviroc-based regimen (75% to 80% range depending on the algorithm used), compared to Trofile (80%) and population sequencing (70%). In conclusion, all four NGS platforms were able to detect minority non-R5 variants at comparable levels suggesting that any NGS-based method can be used to predict HIV-1 coreceptor usage. © 2012 Archer et al.


Gibson R.M.,University Hospital Translational Laboratory | Meyer A.M.,University Hospital Translational Laboratory | Winner D.,University Hospital Translational Laboratory | Archer J.,University of Manchester | And 7 more authors.
Antimicrobial Agents and Chemotherapy | Year: 2014

With 29 individual antiretroviral drugs available from six classes that are approved for the treatment of HIV-1 infection, a combination of different phenotypic and genotypic tests is currently needed to monitor HIV-infected individuals. In this study, we developed a novel HIV-1 genotypic assay based on deep sequencing (DeepGen HIV) to simultaneously assess HIV-1 susceptibilities to all drugs targeting the three viral enzymes and to predict HIV-1 coreceptor tropism. Patient-derived gag-p2/NCp7/p1/p6/ pol-PR/RT/IN- and env-C2V3 PCR products were sequenced using the Ion Torrent Personal Genome Machine. Reads spanning the 3′ end of the Gag, protease (PR), reverse transcriptase (RT), integrase (IN), and V3 regions were extracted, truncated, translated, and assembled for genotype and HIV-1 coreceptor tropism determination. DeepGen HIV consistently detected both minority drug-resistant viruses and non-R5 HIV-1 variants from clinical specimens with viral loads of ≥1,000 copies/ml and from B and non-B subtypes. Additional mutations associated with resistance to PR, RT, and IN inhibitors, previously undetected by standard (Sanger) population sequencing, were reliably identified at frequencies as low as 1%. DeepGen HIV results correlated with phenotypic (original Trofile, 92%; enhanced-sensitivity Trofile assay [ESTA], 80%; TROCAI, 81%; and VeriTrop, 80%) and genotypic (population sequencing/Geno2Pheno with a 10% false-positive rate [FPR], 84%) HIV-1 tropism test results. DeepGen HIV (83%) and Trofile (85%) showed similar concordances with the clinical response following an 8-day course of maraviroc monotherapy (MCT). In summary, this novel all-inclusive HIV-1 genotypic and coreceptor tropism assay, based on deep sequencing of the PR, RT, IN, and V3 regions, permits simultaneous multiplex detection of low-level drug-resistant and/or non-R5 viruses in up to 96 clinical samples. This comprehensive test, the first of its class, will be instrumental in the development of new antiretroviral drugs and, more importantly, will aid in the treatment and management of HIV-infected individuals. Copyright © 2014, American Society for Microbiology. All Rights Reserved.


Gibson R.M.,University Hospital Translational Laboratory | Schmotzer C.L.,University Hospital Translational Laboratory | Schmotzer C.L.,Case Western Reserve University | Quinones-Mateu M.E.,University Hospital Translational Laboratory | Quinones-Mateu M.E.,Case Western Reserve University
Current Infectious Disease Reports | Year: 2014

Given the extreme variability of the human immunodeficiency virus (HIV) and its ability to replicate as complex viral populations, HIV variants with reduced susceptibility to antiretroviral drugs or with specific coreceptor tropism (CCR5 and/or CXCR4) may be present as minority members of the viral quasispecies. The sensitivity of current HIV genotypic or phenotypic assays is limited, and thus, these tests usually fail to detect low-abundance viral variants. Next-generation (deep) sequencing (NGS) produces an enormous amount of information that allows the detection of minority HIV variants at levels unimaginable using standard Sanger sequencing. NGS technologies continue to evolve, opening new and more affordable opportunities to implement this methodology in clinical laboratories, and HIV is not an exception. The ample use of a battery of more effective antiretroviral drugs, together with careful patient monitoring based on HIV resistance testing, has resulted in HIV-infected patients whose disease is usually well-controlled. The vast majority of adherent patients without detectable resistance become virologically suppressed; however, a subset of these patients with undetectable resistance by standard methods may fail antiretroviral therapy, perhaps due to the presence of minority HIV-resistant variants. Novel NGS-based HIV assays with increased sensitivity for identifying low-level drug resistance and/or coreceptor tropism may play an important role in the success of antiretroviral treatments. © 2014 Springer Science+Business Media.


Virk S.M.,Case Western Reserve University | Gibson R.M.,University Hospital Translational Laboratory | Quinones-Mateu M.E.,University Hospital Translational Laboratory | Quinones-Mateu M.E.,Case Western Reserve University | Barnholtz-Sloan J.S.,Case Western Reserve University
PLoS ONE | Year: 2015

Glioblastoma (GBM) is an aggressive, malignant brain tumor typically resulting in death of the patient within one year following diagnosis; and those who survive beyond this point usually present with tumor recurrence within two years (5-year survival is 5%). The genetic heterogeneity of GBM has made the molecular characterization of these tumors an area of great interest and has led to identification of molecular subtypes in GBM. The availability of sequencing platforms that are both fast and economical can further the adoption of tumor sequencing in the clinical environment, potentially leading to identification of clinically actionable genetic targets. In this pilot study, comprised of triplet samples of normal blood, primary tumor, and recurrent tumor samples from three patients; we compared the ability of Illumina whole exome sequencing (ExomeSeq) and the Ion AmpliSeq Comprehensive Cancer Panel (CCP) to identify somatic variants in patient-paired primary and recurrent tumor samples. Thirteen genes were found to harbor variants, the majority of which were exclusive to the ExomeSeq data. Surprisingly, only two variants were identified by both platforms and they were located within the PTCH1 and NF1 genes. Although preliminary in nature, this work highlights major differences in variant identification in data generated from the two platforms. Additional studies with larger samples sizes are needed to further explore the differences between these technologies and to enhance our understanding of the clinical utility of panel based platforms in genomic profiling of brain tumors. © 2015 Virk et al.

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