Low-cost ultra-wide genotyping using Roche/454 pyrosequencing for surveillance of HIV drug resistance.

PloS One
Dawn M DudleyDavid H O'Connor

Abstract

Great efforts have been made to increase accessibility of HIV antiretroviral therapy (ART) in low and middle-income countries. The threat of wide-scale emergence of drug resistance could severely hamper ART scale-up efforts. Population-based surveillance of transmitted HIV drug resistance ensures the use of appropriate first-line regimens to maximize efficacy of ART programs where drug options are limited. However, traditional HIV genotyping is extremely expensive, providing a cost barrier to wide-scale and frequent HIV drug resistance surveillance. We have developed a low-cost laboratory-scale next-generation sequencing-based genotyping method to monitor drug resistance. We designed primers specifically to amplify protease and reverse transcriptase from Brazilian HIV subtypes and developed a multiplexing scheme using multiplex identifier tags to minimize cost while providing more robust data than traditional genotyping techniques. Using this approach, we characterized drug resistance from plasma in 81 HIV infected individuals collected in São Paulo, Brazil. We describe the complexities of analyzing next-generation sequencing data and present a simplified open-source workflow to analyze drug resistance data. From this data, we ...Continue Reading

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Methods Mentioned

BETA
genotyping
PCR
electrophoresis
chip
Transfection
amplicon sequencing

Software Mentioned

LabKey Server
Viroseq
Geneious
LabKey
GS
SW
BWA
Rega

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