Sequencing and Bioinformatics

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ABSTRACT, CORE 4 Next-Generation Sequencing is an essential and fundamental approach in molecular virology. In vitro, it can capture and characterize protein-nucleic interactions and RNA structure. In cell culture and in clinical settings, it can measure rates of viral polymerase error, viral response to small molecule inhibitors or changes in host gene expression. NGS can chart viral evolution, patterns of genome conservation, characterize origins of drug resistance and discover new viruses. As such, NGS has been transformative in our ability to study virus diversity and evolution and is still a burgeoning field. The Sequencing Core comprises a group of experts with experience with the implementation of the wide range of approaches in terms of molecular biology, experimental design, and downstream computational analyses. We will provide expert support and resources to provide Next- Generation Sequencing and downstream computational analyses to the B-HIVE Research Projects. Importantly, a key focus of the core will be to develop and apply approaches that go beyond what is accessible via commercial and university core facilities. We will provide and develop state-of-the-art technologies in close collaboration with the Research Projects that will help provide unique insights into HIV biology and evolution, whilst also providing novel platforms for deployments in other viral systems. A central focus of the B-HIVE is to understand the molecular mechanisms governing HIV entry, integration, particle assembly, and egress. Each of these processes involve profiling the viral and host nucleic acids during the viral lifecycle. The sequencing core will support the entire B-HIVE reflecting stages throughout the viral replication cycle. We will develop single-cell sequencing approaches, leveraging our extensive expertise in this area, to characterize the effects of integration site location, chromatin structure, HIV mutations, and exposure to latency reversing agents (LRAs) on proviral expression. We will apply and advance long-read sequencing technologies using the Oxford Nanopore Technologies MinION platform for both host transcriptomics and full- length HIV genome sequencing to characterize host and virus splicing. We will develop and implement novel approaches in virus genomics to characterize virus sequence diversity in both clinical and experimental settings in support of the main Research Projects. The Sequencing Core has extensive experience developing, deploying and maintaining new computational software and sequencing databases. These will provide an important resource to the B-HIVE centers, as well as allow the rapid dissemination and sharing of capabilities with the broader HIV and virus research community. Finally, we will provide training in each of our respective platforms and technologies in view of disseminating these skills throughout the B-HIVE and to support the development and progression of trainees working with the B-HIVE and its collaborators. 1