title: An international multicenter study on HIV-1 drug resistance testing by 454 ultra-deep pyrosequencing.
abstract: The detection of mutant spectra within the viral quasispecies is critical for therapeutic management of HIV-1 infections. Routine clinical application of ultrasensitive genotyping requires reproducibility and concordance within and between laboratories. The goal of the study was to evaluate a new protocol on HIV-1 drug resistance testing by 454 ultra-deep pyrosequencing (454-UDS) in an international multicenter study. Sixteen blinded HIV-1 subtype B samples were provided for 454-UDS as both RNA and cDNA with viral titers of 88,600-573,000 HIV-1 RNA copies/ml. Eight overlapping amplicons spanning protease (PR) codons 10-99 and reverse transcriptase (RT) codons 1-251 were generated using molecular barcoded primers. 454-UDS was performed using the 454 Life Sciences/Roche GS FLX platform. PR and RT sequences were analyzed using 454 Life Sciences Amplicon Variant Analyzer (AVA) software. Quantified variation data were analyzed for intra-laboratory reproducibility and inter-laboratory concordance. Routine population sequencing was performed using the ViroSeq HIV-1 genotyping system. Eleven laboratories and the reference laboratory 454 Life Sciences sequenced the HIV-1 sample set. Data presented are derived from seven laboratories and the reference laboratory since severe study protocol execution errors occurred in four laboratories leading to exclusion. The median sequencing depth across all sites was 1364 reads per position (IQR=809-2065). 100% of the ViroSeq-reported mutations were also detected by 454-UDS. Minority HIV-1 drug resistance mutations, defined as HIV-1 drug resistance mutations identified at frequencies of 1-25%, were only detected by 454-UDS. Analysis of 10 preselected majority and minority mutations were consistently found across sites. The analysis of drug-resistance mutations detected between 1 and 10% demonstrated high intra- and inter-laboratory consistency in frequency estimates for both RNA and prepared cDNA samples, indicating robustness of the method. HIV-1 drug resistance testing using 454 ultra-deep pyrosequencing results in an accurate and highly reproducible, albeit complex, approach to the analysis of HIV-1 mutant spectra, even at frequencies well below those detected by routine population sequencing.
authors.affiliation: 454 Life Sciences, A Roche Company, Branford, CT, USA.
authors.forename: Birgitte B
authors.initials: BB
authors.lastname: Simen
authors.affiliation: 454 Life Sciences, A Roche Company, Branford, CT, USA.
authors.forename: Michael S
authors.initials: MS
authors.lastname: Braverman
authors.affiliation: National Institute for Infectious Diseases "L. Spallanzani, Rome, Italy.
authors.forename: Isabella
authors.initials: I
authors.lastname: Abbate
authors.affiliation: Janssen Infectious Diseases - Diagnostics bvba, Beerse, Belgium.
authors.forename: Jeroen
authors.initials: J
authors.lastname: Aerssens
authors.affiliation: Centre Jean Perrin/Clermont University, Clermont-Ferrand, France.
authors.forename: Yannick
authors.initials: Y
authors.lastname: Bidet
authors.affiliation: GeT-PlaGe, INRA 0444 LGC, Castanet-Tolosan, France.
authors.forename: Olivier
authors.initials: O
authors.lastname: Bouchez
authors.affiliation: Red Cross Transfusion Service of Upper Austria, Linz, Austria.
authors.forename: Christian
authors.initials: C
authors.lastname: Gabriel
authors.affiliation: INSERM U1043 and Virology Laboratory, CHU Toulouse, Toulouse, France.
authors.forename: Jacques
authors.initials: J
authors.lastname: Izopet
authors.affiliation: Medical University of Graz, Graz, Austria.
authors.forename: Harald H
authors.initials: HH
authors.lastname: Kessler
authors.affiliation: Medical University of Graz, Graz, Austria.
authors.forename: Evelyn
authors.initials: E
authors.lastname: Stelzl
authors.affiliation: Division of Infectious Diseases and Hospital Epidemiology, University Hospital Zurich, University of Zurich, Zurich, Switzerland.
authors.forename: Francesca
authors.initials: F
authors.lastname: Di Giallonardo
authors.affiliation: Functional Genomics Center Zurich, University of Zurich, ETH Zurich, Zurich, Switzerland.
authors.forename: Ralph
authors.initials: R
authors.lastname: Schlapbach
authors.affiliation: Robert Koch-Institute, Berlin, Germany.
authors.forename: Aleksander
authors.initials: A
authors.lastname: Radonic
authors.affiliation: IrsiCaixa AIDS Research Institute, Badalona, Spain.
authors.forename: Roger
authors.initials: R
authors.lastname: Paredes
authors.affiliation: CHU Bordeaux and UMR 5234, MFP Univ. Bordeaux, Bordeaux, France.
authors.forename: Patricia
authors.initials: P
authors.lastname: Recordon-Pinson
authors.affiliation: TIA-National Genomics Platform, Durban, South Africa.
authors.forename: James
authors.initials: J
authors.lastname: Sakwa
authors.affiliation: 454 Life Sciences, A Roche Company, Branford, CT, USA.
authors.forename: Elizabeth P
authors.initials: EP
authors.lastname: St John
authors.affiliation: Roche Applied Science, Penzberg, Germany.
authors.forename: Gudrun G
authors.initials: GG
authors.lastname: Schmitz-Agheguian
authors.affiliation: Division of Infectious Diseases and Hospital Epidemiology, University Hospital Zurich, University of Zurich, Zurich, Switzerland. Electronic address: Karin.Metzner@usz.ch.
authors.forename: Karin J
authors.initials: KJ
authors.lastname: Metzner
authors.affiliation: Institute of Immunology and Genetics, Kaiserslautern, Germany.
authors.forename: Martin P
authors.initials: MP
authors.lastname: Däumer
mesh_terms: D019295:Computational Biology / Q000379:methods; D024882:Drug Resistance, Viral; D060005:Genotyping Techniques / Q000379:methods*; D015658:HIV Infections / Q000821:virology*; D015497:HIV-1 / Q000187:drug effects* / Q000235:genetics* / Q000302:isolation & purification; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D007391:International Cooperation; D008826:Microbial Sensitivity Tests / Q000379:methods; D012984:Software
keywords: 454 ultra-deep pyrosequencing; Drug resistance; HIV-1; Multicenter study; Next-generation sequencing