SUMMARY- CORE B (GENOMICS CORE) Core B (Genomics Core) will provide key support to the mVACS team in Clostridioides difficile genome sequence analysis, antigen design, microbiome sequence analysis, routine statistical analysis, and high content data management. Core B will use advanced genomics-based methods to quantify C. difficile genome composition and genetic variation, and associate these with outcomes. Core B will support analysis of protein structure to allow expression of well-folded domains as mRNAs. Core B will characterize DNA inversion systems and other mechanisms of DNA modification and alterations in gene activity that may impact vaccine efficacy. Core B will also carry out transcriptional profiling on C. difficile isolated from the intestine of vaccinated and immunodeficient mice, to assess how C. difficile responds in vivo to effective and ineffective immune responses. The Aims of Core B are: (1) C. difficile genomics to identify optimal targets for modified mRNA vaccines. Long read and short read data will be merged to generate complete assemblies, as in our previous work. Gene content will be enumerated and compared with clinical outcomes. (2) To assess variation in sequence and abundance of C. difficile vaccine targets. C. difficile isolates are known to encode considerable variation in their surface proteins. In addition, DNA inversion systems encode on-off switches affecting surface proteins and transcription factors, resulting in extensive population heterogeneity. This Aim will thus carry out deep assessment of proteins relevant as modified mRNA vaccine targets, and guide antigen design. (3) Transcriptional profiling and microbiome sequencing for mechanistic investigation. Projects 1-3 and the Clinical Core will generate murine and human fecal samples associated with successful or unsuccessful responses to C. difficile infection; Core B will quantify microbiome composition and metatranscriptomic analysis of C. difficile positive samples by RNA-seq. This core will oversee sequence acquisition and analysis is support of these studies. (4) Data management and archiving. Core C will work with Core A for standard statistical analysis and will archive and manage the diverse data types generated by the U19 team.