Added Value of Deep Sequencing Relative to Population Sequencing in Heavily Pre-Treated HIV-1-Infected Subjects Francisco M. Codon˜ er1*¤, Christian Pou 1 , Alexander Thielen3, Federico Garcı´a4, Rafael Delgado 5 , David Dalmau 6 , Miguel A´ lvarez-Tejado7, Lidia Ruiz1, Bonaventura Clotet1,2, Roger Paredes1,2* 1 Institut de Recerca de la SIDA irsiCaixa-HIVACAT, Badalona, Spain, 2 Unitat VIH, Hospital Universitari Germans Trias I Pujol, Badalona, Spain, 3 Max Planck Institute fu¨ r Informatik, Saarbu¨ cken, Germany, 4 Hospital San Cecilio, Granada, Spain, 5 Hospital 12 de Octubre, Madrid, Spain, 6 Hospital Universitari Mutua Terrassa, Terrassa, Spain, 7 Roche Diagnostics SL, Sant Cugat del Valle`s, Spain Abstract Objective: To explore the potential of deep HIV-1 sequencing for adding clinically relevant information relative to viral population sequencing in heavily pre-treated HIV-1-infected subjects. Methods: In a proof-of-concept study, deep sequencing was compared to population sequencing in HIV-1-infected individuals with previous triple-class virological failure who also developed virologic failure to deep salvage therapy including, at least, darunavir, tipranavir, etravirine or raltegravir. Viral susceptibility was inferred before salvage therapy initiation and at virological failure using deep and population sequencing genotypes interpreted with the HIVdb, Rega and ANRS algorithms. The threshold level for mutant detection with deep sequencing was 1%. Results: 7 subjects with previous exposure to a median of 15 antiretrovirals during a median of 13 years were included. Deep salvage therapy included darunavir, tipranavir, etravirine or raltegravir in 4, 2, 2 and 5 subjects, respectively. Self- reported treatment adherence was adequate in 4 and partial in 2; one individual underwent treatment interruption during follow-up. Deep sequencing detected all mutations found by population sequencing and identified additional resistance mutations in all but one individual, predominantly after virological failure to deep salvage therapy. Additional genotypic information led to consistent decreases in predicted susceptibility to etravirine, efavirenz, nucleoside reverse transcriptase inhibitors and indinavir in 2, 1, 2 and 1 subject, respectively. Deep sequencing data did not consistently modify the susceptibility predictions achieved with population sequencing for darunavir, tipranavir or raltegravir. Conclusions: In this subset of heavily pre-treated individuals, deep sequencing improved the assessment of genotypic resistance to etravirine, but did not consistently provide additional information on darunavir, tipranavir or raltegravir susceptibility. These data may inform the design of future studies addressing the clinical value of minority drug-resistant variants in treatment-experienced subjects. Citation: Codo n˜er FM, Pou C, Thielen A, Garc´ıa F, Delgado R, et al. (2011) Added Value of Deep Sequencing Relative to Population Sequencing in Heavily Pre- Treated HIV-1-Infected Subjects. PLoS ONE 6(5): e19461. doi:10.1371/journal.pone.0019461 Editor: Esper Georges Kallas, University of Sao Paulo, Brazil Received January 17, 2011; Accepted March 30, 2011; Published May 13, 2011 Copyright: 2011 Codon˜ er et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was supported by the CDTI (Centro para el Desarrollo Tecnolo´ gico Industrial), Spanish Ministry of Science and Innovation (IDI-20080843) and ‘CHAIN, Collaborative HIV and Anti-HIV Drug Resistance Network’, Integrated Project no. 223131, funded by the European Commission Framework 7 Program. FMC was supported by Marie Curie European Reintegration Grant number 238885, ‘HIV Coevolution’, funded by the European Commission Framework 7 Program. Miguel A´lvarez-Tejado works for Roche Diagnostics, S.L. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: F. Garc´ıa has been a consultant on advisory boards, or has participated in speakers’ bureaus, with Roche, Boehringer-Ingelheim, BMS, GSK, Gilead, Janssen, Merck and Pfizer. R. Delgado has received consulting fees and grant support from Abbott, BMS, Gilead and Roche. D. Dalmau has been a consultant on advisory boards, has participated in speakers’ bureaus, or has conducted clinical trials with Roche, Boehringer-Ingelheim, Abbott, BMS, GSK, Gilead, Tibotec, Janssen, Merck and Pfizer. M. A´ lvarez-Tejado works for Roche Diagnostics S.L., Spain, which commercializes the 454 sequencing technology in Spain. B. Clotet has been a consultant on advisory boards, has participated in speakers’ bureaus, or has conducted clinical trials with Roche, Boehringer-Ingelheim, Abbott, BMS, GSK, Gilead, Tibotec, Janssen, Merck, Pfizer, Siemens, Monogram Biosciences, and Panacos. R. Paredes has received consulting fees from Pfizer and grant support from Pfizer, Siemens, Merck and Boehringer Ingelheim. This does not alter the authors’ adherence to all the PLoS ONE policies on sharing data and materials. F.M. Codon˜ er, C. Pou, A. Thielen, and L. Ruiz report no conflicts of interest relevant to this article. No other potential conflict of interest relevant to this article was reported. * E-mail: fcodoner@gmail.com (FMC); rparedes@irsicaixa.es (RP) ¤ Current address: Lifesequencing, S.L., Valencia, Spain Introduction The rate of virological failure of the 3 original drug classes is low, but not negligible, and does not appear to diminish over time from starting antiretroviral therapy.[1] If this trend continues, many patients will require newer drugs to maintain viral suppression and accurate resistance tests will be needed to guide clinical management. Deep HIV-1 sequencing (454 Life Sciences/ PLoS ONE | www.plosone.org 1 May 2011 | Volume 6 | Issue 5 | e19461