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A semester-long course covering best practices for the analysis of high-throughput sequencing data from gene expression (RNA-seq) studies, with a primary focus on empowering students to be independent in the use of lightweight and open-source software using the R programming language and the Bioconductor suite of packages. This course follows a hybrid format in which online lectures are paired with in-person labs where students participate in hands-on, live coding exercises using real ‘omic datasets. The course is focused on datasets and topics central to infectious disease research, immunology, and One-Health, but the concepts and approaches covered are applicable to any genomic study.