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Zagordi O.,University of Zurich | Daumer M.,Institute of Immunology and Genetics | Beisel C.,ETH Zurich | Beerenwinkel N.,ETH Zurich | Beerenwinkel N.,Swiss Institute of Bioinformatics
PLoS ONE | Year: 2012

Recent advancements of sequencing technology have opened up unprecedented opportunities in many application areas. Virus samples can now be sequenced efficiently with very deep coverage to infer the genetic diversity of the underlying virus populations. Several sequencing platforms with different underlying technologies and performance characteristics are available for viral diversity studies. Here, we investigate how the differences between two common platforms provided by 454/Roche and Illumina affect viral diversity estimation and the reconstruction of viral haplotypes. Using a mixture of ten HIV clones sequenced with both platforms and additional simulation experiments, we assessed the trade-off between sequencing coverage, read length, and error rate. For fixed costs, short Illumina reads can be generated at higher coverage and allow for detecting variants at lower frequencies. They can also be sufficient to assess the diversity of the sample if sequences are dissimilar enough, but, in general, assembly of full-length haplotypes is feasible only with the longer 454/Roche reads. The quantitative comparison highlights the advantages and disadvantages of both platforms and provides guidance for the design of viral diversity studies. © 2012 Zagordi et al. Source

Verheyen J.,University of Cologne | Schweitzer F.,University of Cologne | Harrer E.G.,Friedrich - Alexander - University, Erlangen - Nuremberg | Knops E.,University of Cologne | And 7 more authors.
Antiviral Therapy | Year: 2010

Introduction: HIV type-1 (HIV-1) protease (PR) and cleavage site (CS) mutations accumulate in protease-inhibitor-resistant isolates. HIV-1 CS mutation 431V is the most frequent treatment-associated CS mutation; however, little is known about its origin in treatment-naive HIV-1 isolates. Recently, it has been shown that the CS mutation 431V is located within the human leukocyte antigen (HLA)-B*13-restricted cytotoxic T- lymphocyte (CTL) epitope RQANFLGKI (RI9). Therefore, we investigated whether the presence of CS mutation 431V might additionally be related to immune escape. Methods: CTL recognition of RI9 and of RI9 variants carrying the 431V or the 436R mutation was analysed by ELISPOT in nine HLA-B*13-positive HIV-1-infected patients. Treatment-naive HIV-1-infected patients with primary drug-resistant HIV-1 isolates (n=58) or carrying 431V (n=4) were genotyped for HLA class I alleles. Results: ELISPOT analysis showed different patterns of CTL recognition of RI9. CS mutation 431V could abrogate recognition by RI9-specific CTL in a subgroup of patients. Nevertheless, in our study, the occurrence of 431V in treatment-naive HIV-1 without primary drug resistance could not be explained by HLA-B*13- mediated immune selection. In patients with primary drug-resistant HIV-1 isolates, the frequency of HLA-B*13 was not increased and HLA-B*13 did not correlate with CS mutations 436R or 431V. Conclusions: HIV-1 CS mutation 431V can abrogate CTL recognition, indicating interactions between development of drug resistance and the CTL response. However, we could not find evidence that the presence of 431V in treatment-naive HIV-1 isolates with and without primary drug resistance is related to immune selection by HLA-B*13 or other HLA class I alleles. ©2010 International Medical Press. Source

Swenson L.C.,British Columbia Center for Excellence in | Daumer M.,Institute of Immunology and Genetics | Paredes R.,Autonomous University of Barcelona
Current Opinion in HIV and AIDS | Year: 2012

PURPOSE OF REVIEW: Deep sequencing of the V3 region of the HIV envelope gene can detect minority non-R5 variants in patients with high sensitivity and specificity. As next-generation sequencing approaches have matured, the clinical utility of deep sequencing for HIV tropism has entered the clinic. Accurate and sensitive tropism testing is essential for successful treatment with the CCR5 antagonist class of antiretrovirals. RECENT FINDINGS: This review will focus on five aspects of next-generation sequencing for assessing HIV tropism: some background on the necessity of deep sequencing versus other tropism methods; the methodological process of 454 sequencing and analysis; other next-generation sequencing technologies; the diagnostic performance of deep sequencing relative to other tropism assays; and the use of deep sequencing in clinical practice. SUMMARY: This method has emerged quickly as both a research and clinical tool because of its high concordance with commonly used phenotypic tropism assays and its ability to predict virological response to CCR5 antagonist-containing regimens. © 2012 Wolters Kluwer Health | Lippincott Williams & Wilkins. Source

Knops E.,University of Cologne | Daumer M.,Institute of Immunology and Genetics | Awerkiew S.,University of Cologne | Kartashev V.,Rostov State Medical University | And 6 more authors.
Journal of Antimicrobial Chemotherapy | Year: 2010

Objectives: To analyse HIV Gag cleavage site (CS) and non-CS mutations in HIV non-B isolates from patients failing antiretroviral therapy. Patients and methods: Twenty-one HIV isolates were obtained from patients infected with HIV subtype G during an outbreak in Russia 20 years ago. Most patients were failing antiretroviral therapy when genotyping was performed. Results: HIV Gag CS mutations accumulated in protease inhibitor (PI)-resistant HIV isolates and were correlated with the presence of three or more PI resistance mutations. Only 1 of 11 HIV isolates carrying major protease mutations did not harbour treatment-associated CS mutations. Natural polymorphism 453T, often found in HIV non-B subtypes, seems to favour the selection of CS mutation 453I rather than treatment-associated CS mutation 453L. Resistance-associated non-CS mutations (123E and 200I) were also observed in PI-resistant clinical isolates. Non-CS mutations in the frameshift-regulating site, which controls the synthesis of Gag-Pol, did not affect frameshift efficiency in dual luciferase assays. Of note, one of four HIV isolates from patients failing PI therapies without protease mutations harboured Gag mutations associated with PI resistance (123E and 436R) and reverse transcriptase inhibitor mutations conferring resistance to the backbone drug. Conclusions: HIV Gag CS mutations commonly occurred in HIV isolates from patients failing PI therapies and natural polymorphisms at the same position influence their emergence. Non-CS mutations previously associated with PI resistance were also observed in clinical isolates. Gag mutations might indicate the evolution of PI resistance even in the absence of protease mutations. © The Author 2010. Source

Thielen A.,Max Planck Institute for Informatics | Thielen A.,Institute of Immunology and Genetics | Lengauer T.,Max Planck Institute for Informatics
Intervirology | Year: 2012

Inferring HIV-1 coreceptor usage from a genotype is becoming more and more important for the appropriate treatment of long-term patients. While results are already encouraging where standard bulk-nucleic acid sequencing methods are used, they are limited with respect to the detection of minor variants. In contrast, next-generation sequencing methods (ultradeep sequencing, pyrosequencing) are capable of sequencing virus quasispecies at very low quantities. However, as well as being very expensive, these methods generate vast amounts of data such that sequence analysis has to be automated by computer assistance. Here, we describe the geno2pheno[454] system which handles all processing and prediction steps involved in the prediction of coreceptor usage from massively parallel sequencing data. The system is split into a JAVA preprocessor which is run locally on the client side and a Web server which generates the prediction results. Predictions are based on the same prediction method as used in the geno2pheno[coreceptor] tool. Copyright © 2012 S. Karger AG, Basel. Source

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