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.

Figure 1.

Amplicons querying positions of HIV drug resistance mutations in the PR and RT-coding regions. The HIV genetic map is shown at the top. Below is shown an expanded version of the segment of pol encoding PR and part of RT. At the bottom is shown the location of the amplicons used to interrogate positions of potential drug resistance mutations.

Figure 2.

The DNA bar coding system. Primers are shown diagrammatically, illustrating the placement of the DNA bar code between the 454 primers (A and B) and the HIV-complementary sequences. An example of a primer sequence is shown at the bottom. DNA 5′ ends in the primers are shown as the black balls.

Figure 3.

Amplification of HIV subtype C using the pan-HIV primer set. The figure shows products of RT-PCR reactions analyzed by agarose gel electrophoresis and ethidium bromide staining. The amplicons extend from the 5′ side of the PR-coding region (left) to an internal region of the RT-coding region (right), thereby covering all the known positions of drug resistance mutations in the PR and RT-coding regions (primer sequences are in Supplementary Table 1). The marker ladder has ‘steps’ of 100 bp. The position of the 500 bp fragment is marked.

Figure 4.

Summary of the drug resistance alleles detected. Results are shown for PR (top two grids) and RT (bottom two grids). The seven HIV samples studied are labeled to the left of each grid. The amino acid positions of drug-resistant mutations are marked at the top of the grid, labeled DRM for drug resistance mutation. Black tiles in the grid indicate positions that were significantly enriched for drug resistance mutations in the pyrosequencing data after correction for multiple comparisons. Raw counts are available in Supplementary Table 2. Black tiles with asterisks indicate positions that were enriched in drug resistance mutations in the pyrosequencing data that were not identified by a conventional genotyping method (Viroseq HIV Genotyping System v 2.0). The ‘i’ at RT position 69 denotes the insertions at this position that are known to confer drug resistance.