title: DNA bar coding and pyrosequencing to identify rare HIV drug resistance mutations.
abstract: Treatment of HIV-infected individuals with antiretroviral agents selects for drug-resistant mutants, resulting in frequent treatment failures. Although the major antiretroviral resistance mutations are routinely characterized by DNA sequencing, treatment failures are still common, probably in part because undetected rare resistance mutations facilitate viral escape. Here we combined DNA bar coding and massively parallel pyrosequencing to quantify rare drug resistance mutations. Using DNA bar coding, we were able to analyze seven viral populations in parallel, overall characterizing 118 093 sequence reads of average length 103 bp. Analysis of a control HIV mixture showed that resistance mutations present as 5% of the population could be readily detected without false positive calls. In three samples of multidrug-resistant HIV populations from patients, all the drug-resistant mutations called by conventional analysis were identified, as well as four additional low abundance drug resistance mutations, some of which would be expected to influence the response to antiretroviral therapy. Methods for sensitive characterization of HIV resistance alleles have been reported, but only the pyrosequencing method allows all the positions at risk for drug resistance mutations to be interrogated deeply for many HIV populations in a single experiment.
authors.affiliation: Department of Microbiology, University of Pennsylvania School of Medicine, 3610 Hamilton Walk, Philadelphia, PA 19104-6076, USA.
authors.forename: Christian
authors.initials: C
authors.lastname: Hoffmann
authors.forename: Nana
authors.initials: N
authors.lastname: Minkah
authors.forename: Jeremy
authors.initials: J
authors.lastname: Leipzig
authors.forename: Gary
authors.initials: G
authors.lastname: Wang
authors.forename: Max Q
authors.initials: MQ
authors.lastname: Arens
authors.forename: Pablo
authors.initials: P
authors.lastname: Tebas
authors.forename: Frederic D
authors.initials: FD
authors.lastname: Bushman
mesh_terms: D000483:Alleles; D019380:Anti-HIV Agents / Q000627:therapeutic use*; D004279:DNA, Viral / Q000737:chemistry; D003627:Data Interpretation, Statistical; D024882:Drug Resistance, Viral / Q000235:genetics; D006678:HIV / Q000187:drug effects / Q000235:genetics*; D015658:HIV Infections / Q000188:drug therapy; D006801:Humans; D009154:Mutation; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA / Q000379:methods*
chemical_list: D019380:Anti-HIV Agents; D004279:DNA, Viral