title: A Follow-Up of the Multicenter Collaborative Study on HIV-1 Drug Resistance and Tropism Testing Using 454 Ultra Deep Pyrosequencing.
abstract: BACKGROUND
  Ultra deep sequencing is of increasing use not only in research but also in diagnostics. For implementation of ultra deep sequencing assays in clinical laboratories for routine diagnostics, intra- and inter-laboratory testing are of the utmost importance.
  
  
  METHODS
  A multicenter study was conducted to validate an updated assay design for 454 Life Sciences' GS FLX Titanium system targeting protease/reverse transcriptase (RTP) and env (V3) regions to identify HIV-1 drug-resistance mutations and determine co-receptor use with high sensitivity. The study included 30 HIV-1 subtype B and 6 subtype non-B samples with viral titers (VT) of 3,940-447,400 copies/mL, two dilution series (52,129-1,340 and 25,130-734 copies/mL), and triplicate samples. Amplicons spanning PR codons 10-99, RT codons 1-251 and the entire V3 region were generated using barcoded primers. Analysis was performed using the GS Amplicon Variant Analyzer and geno2pheno for tropism. For comparison, population sequencing was performed using the ViroSeq HIV-1 genotyping system.
  
  
  RESULTS
  The median sequencing depth across the 11 sites was 1,829 reads per position for RTP (IQR 592-3,488) and 2,410 for V3 (IQR 786-3,695). 10 preselected drug resistant variants were measured across sites and showed high inter-laboratory correlation across all sites with data (P<0.001). The triplicate samples of a plasmid mixture confirmed the high inter-laboratory consistency (mean% ± stdev: 4.6 ±0.5, 4.8 ±0.4, 4.9 ±0.3) and revealed good intra-laboratory consistency (mean% range ± stdev range: 4.2-5.2 ± 0.04-0.65). In the two dilutions series, no variants >20% were missed, variants 2-10% were detected at most sites (even at low VT), and variants 1-2% were detected by some sites. All mutations detected by population sequencing were also detected by UDS.
  
  
  CONCLUSIONS
  This assay design results in an accurate and reproducible approach to analyze HIV-1 mutant spectra, even at variant frequencies well below those routinely detectable by population sequencing.
authors.affiliation: 454 Life Sciences, A Roche Company, Branford, CT, United States of America.
authors.forename: Elizabeth P
authors.initials: EP
authors.lastname: St John
authors.affiliation: 454 Life Sciences, A Roche Company, Branford, CT, United States of America.
authors.forename: Birgitte B
authors.initials: BB
authors.lastname: Simen
authors.affiliation: 454 Life Sciences, A Roche Company, Branford, CT, United States of America.
authors.forename: Gregory S
authors.initials: GS
authors.lastname: Turenchalk
authors.affiliation: 454 Life Sciences, A Roche Company, Branford, CT, United States of America.
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: Plateforme Génomique Toulouse/Laboratoire Génétique Cellulaire, Toulouse, France.
authors.forename: Olivier
authors.initials: O
authors.lastname: Bouchez
authors.affiliation: Blutzentrale Linz, Linz, Austria.
authors.forename: Christian
authors.initials: C
authors.lastname: Gabriel
authors.affiliation: INSERM U563, Toulouse, France.
authors.forename: Jacques
authors.initials: J
authors.lastname: Izopet
authors.affiliation: Robert Koch-Institute, Berlin, Germany.
authors.forename: Karolin
authors.initials: K
authors.lastname: Meixenberger
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: Institut de Recerca de la SIDA-IrsiCaixa, Badalona, Spain.
authors.forename: Roger
authors.initials: R
authors.lastname: Paredes
authors.affiliation: Technology Innovation Agency-National Genomics Platform, Durban, South Africa.
authors.forename: James
authors.initials: J
authors.lastname: Sakwa
authors.affiliation: Roche Applied Science, Penzberg, Germany.
authors.forename: Gudrun G
authors.initials: GG
authors.lastname: Schmitz-Agheguian
authors.affiliation: Institute of Immunology and Genetics, Kaiserslautern, Germany.
authors.forename: Alexander
authors.initials: A
authors.lastname: Thielen
authors.affiliation: Roche Applied Science, Penzberg, Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: Victor
authors.affiliation: Division of Infectious Diseases and Hospital Epidemiology, University Hospital Zurich, University of Zurich, Zurich, Switzerland.|Institute of Medical Virology, University of Zurich, Zurich, Switzerland.
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: D000596:Amino Acids / Q000235:genetics; D003299:Cooperative Behavior; D024882:Drug Resistance, Viral / Q000235:genetics*; D005500:Follow-Up Studies; D015497:HIV-1 / Q000235:genetics*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D009154:Mutation / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D015203:Reproducibility of Results; D018523:Tropism / Q000235:genetics*
chemical_list: D000596:Amino Acids