The Viral Evolution Core was established in 2009 with a mission to provide expertise in specialized sequencing techniques, molecular cloning, and viral evolution analyses to support the AIDS and Cancer Virus Program, National Cancer Institute, National Institutes of Health, and extramural investigators in conducting a broad range of AIDS-research studies, including HIV-cure research, viral reservoir identification/elimination, viral transmission/dissemination, and viral adaptation.
We seek to improve the overall understanding of viral evolution, specifically capitalizing on the unique advantages afforded by nonhuman primate models of infection with SIV and SIV-based chimeric viruses. We also sequence virus from individuals living with HIV for cure/reservoir research, studies identifying viral compartmentalization, and monitoring viral adaptation over time.
Our core has established critical infrastructure and essential personnel to provide key genetic insights into viral evolution for a diverse group of scientists who might lack the capability and expertise to perform such analyses in their individual laboratories. Our collaborators include researchers from both within the AIDS and Cancer Virus Program as well as intramural and extramural labs.
Sequencing provides insight into the evolution, dynamics, and characterization of AIDS viruses
The viral genome holds a wealth of information that can often only be accessed by performing sequencing analysis. By sequencing, we glean data on a virus’s tropism, its ability to escape immune pressures or generate drug resistance, its intactness, its phylogeny, and its integration site in the host genome, to name a few. Insights learned by delving into these aspects of viral characterization provide clues into how to best treat and, ultimately, cure HIV.
Customized sequencing and analytical services
The Viral Evolution Core provides a range of fully optimized sequencing and analytical services to its collaborators but also will generate customized protocols for either single-genome amplification paired with Sanger sequencing or Illumina-based, next-generation sequencing based on the objectives of the study at hand.
One of the major benefits that we offer is our close collaboration with a computational biology team who are able to generate scripted pipelines for the various analyses necessary to translate our sequencing data from millions of single reads into a comprehensive and comprehendible output.
Sequencing for a multi-laboratory Gates Consortium
This consortium seeks to determine the anatomical origins of the rebound-competent viral reservoir and identify a marker of infected cells by quantifying and sequencing the virus in all anatomical compartments throughout the body, followed by extensive transcriptomic analysis to capture any differential gene expression in infected cells.
Our core is tasked with providing all the sequencing services for this landmark project.
Collaboration Opportunities
We work with investigators through various partnership mechanisms, including Material Transfer Agreements (MTAs), FNL Cooperative Research and Development Agreements (FNL CRADAs), Technical Service Agreements (TSAs), and Collaboration Agreements.
Contact Christine Fennessey to inquire about and engage the core’s services.
Discovering novel aspects of HIV biology through sequencing
We employ advanced sequencing techniques to support a broad range of HIV/SIV research protocols.
This may entail sequencing of bulk DNA/RNA, single genome amplification (SGA) which allows sequencing of individual templates from a mixed population, or next-generation sequencing (NGS) which can provide ultra-deep analysis of the viral population in a single region of the genome or a broad view of the genetic diversity present by deep sequencing small amplicons across the whole genome, or a combination of all three approaches.
Our services have provided crucial data that drive study development and conclusions for our collaborators.
Our capabilities and specializations
Single-genome amplification and sequencing
We perform PCR and Sanger sequencing on limiting dilution single viral templates to identify genetic variations within a population at a deeper resolution than standard bulk sequencing.
This approach provides three fundamental advantages over standard bulk PCR and sequencing. First, it prevents in vitro recombination, which ensures accurate genetic linkage across the entire amplicon. Second, it excludes Taq based errors during PCR. Third, it allows for the proportional representation of the viral population. These critical advantages have proven invaluable in many studies since the technique was developed over a decade ago.
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Single-gene or full-genome amplification
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Intactness/hypermutation characterization
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Identification of drug resistance/CTL escape
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Monitoring viral evolution in response to vaccine/bNAbs
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Phylogenetic analysis
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Custom protocols developed as requested
Illumina-based next-generation sequencing
We provide specialized sequence analysis of barcoded viruses developed by the Retroviral Evolution Section, but also apply Illumina technology to a range of other viral targets.
We have used this approach in numerous different configurations, including CTL escape and drug resistance mutations, nef open/closed assessment, variations in the primer binding site, measuring polymorphisms in V3-V4 and V1-V3 in the env gene, integration site analysis, and variability in other discreet regions of the genome.
However, with the widespread adoption of the barcoded virus for various nonhuman primate studies, the most common usage of this technology is for the identification and quantification of viral barcode lineages from plasma and tissues. This type of sequencing allows an unprecedented identification of minor variants that would likely never be detected if using SGA or standard whole genome NGS approaches.
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SIV/HIV/SHIV/stHIV barcode
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CTL escape sites
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V2V3/α4β7 sequencing
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Integration site analysis
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Transcriptomic library sequencing
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Custom amplicons developed as requested