The new deep sequencing methods offer tremendously improved means of characterizing genetic diversity. The Sequencing and Viral Evolution Core, under the direction of Dr. F. Bushman, will perform deep sequencing to support the IPCP team. The core will analyze viral populations in the presence and absence of therapy to monitor the effects of treatment, characterize the effects of zinc-finger nucleases on genomic DMA, and quantify lentiviral vector integration sites to monitor for possible genotoxicity. The Bushman laboratory has published extensively on applications of deep sequencing, and will adapt these methods to support the proposed projects. The effects of the gag and pol epitope targeting will be assessed by quantifying longitudinal changes in viral quasispecies that accumulate selectively in the presence of pressure from therapy. For HIV viruses replicating in cells with the CXCR4 and CCR5 knockouts, deep sequencing will be used to characterize viral envelope sequences present prior to therapy and their evolution under therapy. Deep sequencing will be used to characterize the genomic structure at the site of gene deletion mediated by zinc finger nucleases and scan for possible associated chromosomal abnormalities. Deep sequencing will also be used to follow integration site selection by lentiviral vectors, and monitor for possible genotoxicity due to vector integration.
Our specific aims are as follows:
Specific Aim 1 : DNA barcoding and pyrosequencing for analysis of viral mutations in response to gag and pol epitope targeting (Projects 1 and 2), and zinc finger mediated CXCR4 and CCR5 knockout in CD4 T cells (Projects 2 and 3).
Specific Aim 2 : Longitudinal analysis of lentiviral vector integration sites using pyrosequencing (Projects 1 and 2).
The new 454/Roche pyrosequencing method allows up to 100,000,000 bases of DNA sequence to be determined in a single one day run. The core will use this method to evaluate the effectiveness of therapy and monitor for possible adverse events.
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