Nucleic Acids Research Advance Access originally published online on June 18, 2007
Nucleic Acids Research 2007 35(13):e91; doi:10.1093/nar/gkm435
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Nucleic Acids Research, 2007, Vol. 35, No. 13 e91
© 2007 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
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DNA bar coding and pyrosequencing to identify rare HIV drug resistance mutations
1Department of Microbiology and 2Division of Infectious Diseases, Department of Medicine, University of Pennsylvania School of Medicine, 3610 Hamilton Walk, Philadelphia, PA 19104-6076 and 3Department of Pediatrics, Washington University School of Medicine, One Children's Place, St Louis, MO 63110, USA
*To whom correspondence should be addressed. Tel: 1 (215) 573 8732; Fax: 1 (215) 573 4856; Email: bushman{at}mail.med.upenn.edu
Received March 2, 2007. Revised March 2, 2007. Accepted May 15, 2007.
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.
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