HIV-1 nucleotide mixture detection in the virco(®)TYPE HIV-1 genotyping assay: a comparison between Sanger sequencing and 454 pyrosequencing.

HIV-1 Protease (PR) and Reverse Transcriptase (RT) genotyping is well established for the management of antiretroviral (ARV) drug therapy, as it is able to detect gene mutations encoding resistance to ARV compounds or drug classes, that are associated with reduced drug susceptibility (i.e. phenotype). A correct phenotypic interpretation from the derived PR-RT genotype (i.e. virtual phenotype), requires a well characterized geno-phenotype correlative database and appropriate statistical predictive models. The applicability of the virtual phenotype for the patient, will, however, not only depend on the accuracy of the statistical models and the database they rely on, but also depend largely on the sequence information that is provided. Since HIV-1 evolves as a complex of closely related but non-identical viral genomes (i.e. quasispecies) it is crucial that the sequencing method used, is able to characterize most of the genetic mixtures that make up the different quasispecies within a single patient. US regulatory agencies require that developers of HIV-1 genotyping assays, determine and report the HIV-1 mixture detection level of their assay. Hence, the mixture scoring sensitivity of the population-based Sanger sequencing method, along with the defined mixture scoring rules, used to drive the virco(®)TYPE HIV-1 virtual phenotype, was investigated by comparing it to the 454 pyrosequencing technique, which is able to generate the complete viral population sequence. To this end the PR-RT coding sequence of 20 clinical isolates was determined by both sequencing methodologies. The genotyping assay which feeds the virco(®)TYPE HIV-1 virtual phenotype was able to call automatically 97.5% (i.e. 268 mixtures) and 95.3% (i.e. 326 mixtures) of the mixtures that were present between 25 and 75% and between 20 and 80% in the viral population, as detected by 454. From the not called mixtures, all but one did present a mixture sequence in the Sanger DNA chromatograms, however, with a peak surface area for the second peak that was below the threshold setting for automatic mixture calling in the basecaller software (i.e. 25%). Viral loads ranged from 470 to 629,000 copies/mL and exerted no effect on the mixture calling relationship between both sequencing methodologies (R(2)=0.92). In some occasions (i.e. 55 mixtures) the genotyping assay would detect automatically mixtures that were present below 20% in the viral population, when measured by 454. Hence, the mixture scoring sensitivity of the automated high throughput virco(®)TYPE HIV-1 genotyping assay is currently set at 97.5% and 95.3%, for mixtures present at 25 and 20% in the viral population and may identify occasionally mutations that are present at lower frequencies. These findings were not influenced by the viral load of the examined samples.