A Follow-Up of the Multicenter Collaborative Study on HIV-1 Drug Resistance and Tropism Testing Using 454 Ultra Deep Pyrosequencing
Saint John, Elizabeth P. (454 Life Sciences (Brandford, Estats Units d'Amèrica))
Simen, Birgitte B. (454 Life Sciences (Brandford, Estats Units d'Amèrica))
Turenchalk, Gregory S. (454 Life Sciences (Brandford, Estats Units d'Amèrica))
Braverman, Michael S. (454 Life Sciences (Brandford, Estats Units d'Amèrica))
Abbate, Isabella (National Institute for Infectious Diseases (Roma, Itàlia))
Aerssens, Jeroen (Janssen Infectious Diseases (Beerse, Bèlgica))
Bouchez, Olivier (Plateforme Génomique Toulouse/Laboratoire Génétique Cellulaire (Toulouse, França))
Gabriel, Christian (Blutzentrale Linz (Àustria))
Izopet, Jacques (INSERM U56 (Toulouse, França))
Meixenberger, Karolin (Robert Koch-Institute (Berlin, Alemanya))
Di Giallonardo, Francesca (University of Zurich)
Schlapbach, Ralph (University of Zurich)
Paredes, Roger (Institut Germans Trias i Pujol. Institut de Recerca de la Sida IrsiCaixa)
Sakwa, James (Technology Innovation Agency-National Genomics Platform (Durban, Sud-àfrica))
Schmitz-Agheguian, Gudrun G. (Roche Applied Science (Penzberg, Alemanya))
Thielen, Alexander (Institute of Immunology and Genetics (Kaiserslautern, Alemanya))
Victor, Martin (Roche Applied Science (Penzberg, Alemanya))
Metzner, Karin J. (University of Zurich)
Däumer, Martin (Institute of Immunology and Genetics (Kaiserslautern, Alemanya))
454 HIV-1 Alpha Study Group

Date:	2016
Abstract:	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. 5A 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. 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. 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.
Rights:	Aquest document està subjecte a una llicència d'ús Creative Commons. Es permet la reproducció total o parcial, la distribució, la comunicació pública de l'obra i la creació d'obres derivades, fins i tot amb finalitats comercials, sempre i quan es reconegui l'autoria de l'obra original.
Language:	Anglès
Document:	Article ; recerca ; Versió publicada
Published in:	PloS one, Vol. 11 Núm. 1 (January 2016) , ISSN 1932-6203

DOI: 10.1371/journal.pone.0146687
PMID: 26756901

17 p, 2.2 MB

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Research literature > UAB research groups literature > Research Centres and Groups (research output) > Health sciences and biosciences > Institut d'Investigació en Ciencies de la Salut Germans Trias i Pujol (IGTP)
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