ACVP-05: Virus Genetic Analysis from Cell-Free Plasma, Virally Infected Cells or Tissues and Cultured Supernatant Via Single Genome Amplification and Direct Sequencing

The Viral Evolution Core within the AIDS and Cancer Virus Program will extract viral RNA/DNA from cell-free or cell-associated samples. Complementary (cDNA) will be generated as needed, and cDNA or DNA will be diluted to a single copy prior to nested polymerase chain reaction (PCR) spanning the viral gene of interest. Following PCR, amplicons are selected by gel electrophoresis and sequenced using Sanger sequencing. To ensure only a single molecule is present in positive reactions, PCR is performed with many replicates, and using the Poisson distribution, the proportion of positive to negative reactions indicates the probability of a single template. Confirmation of single-molecule PCR is obtained by directly sequencing PCR products and excluding amplicons with evidence of more than one template. The Viral Evolution Core will provide comprehensive sequence analysis, including enumerating transmitted/founder genomes, phylogenetic tree construction, and genetic comparisons of diversity. Relevant data will be provided electronically, and a report with interpretation/conclusions will be provided. Sufficient basic consultation with a subject matter expert is included with the service.