Next-generation sequencing of dried blood spot specimens: a novel approach to HIV drug-resistance surveillance.

摘要: BACKGROUND HIV drug-resistance (DR) surveillance in resource-limited settings can be performed using dried blood spots (DBS) because of ease collection, transportation and storage. Analysis pooled specimens on next-generation sequencing (NGS)-based platforms, such as the 454 pyrosequencing, is an efficient method for determining DR rates. In this study, we conducted DBS NGS identified minority variants individual patients. METHODS A total 48 extracts from study Mexico City were re-amplified primers tagged with multiplex identifiers, pyrosequenced. Consensus sequences generated each specimen mixtures at positions where >20% reads contained a variant. Individual consensus then analysed mutations compared those derived Sanger sequencing. RESULTS pyrosequencing (TPP) highly concordant genotypes matching plasma (99.21% 99.51%, respectively). An exception was M184I mutation only detected TPP frequency 20.4%. Multiple had variant below 20% mixture threshold. CONCLUSIONS effective surveillance. genotyping results cost savings 40% over conventional in-house methods. The effect low-abundance mutations, undetectable by methods, remains to determined. This technology might applied any (plasma/serum) also used other diagnostic assays DNA required.