Summary/Abstract Tremendous progress on cancer has been made at the molecular level over the past decade, largely due to the broad application of high throughput, large-scale bulk whole genome, exome and RNA sequencing. In particular, the discovery of numerous medium to high-penetrance drivers, characterization of pathogenic germline variants, and the revelation of many-to-many relationships of genes and pathways, have brought a fuller view of the combinatorial complexity of cancer. Indeed, newer technologies, like single-cell and spatial genomics methods, are now augmenting bulk sequence data to power deeper studies of cancer dynamics, such as heterogeneity, evolution, and interaction with the microenvironment. The current view is that such advanced data, augmented by improved bioinformatics analysis tools and larger, well-curated cohorts will enable medicine to push beyond statistical descriptions toward a genuine deterministic understanding of cancer. Toward this goal, our proposal seeks to extend and apply established bioinformatics systems to integrate the above technologies and leverage our broad range of capabilities and to support the NCI Genomic Characterization Network (NCI-GCN) and Center for Cancer Genomics (CCG) via three specific aims: (1) annotating and interpreting coding and non-coding somatic and germline alterations, (2) characterizing tumor cell populations, evolution, and the tumor microenvironment, and (3) unlocking biological and clinical insights at both the individual and cross-cancer (Pan-Cancer) levels to discern basic themes across the major human cancers. Our approach involves fluencies in four areas of core competence outlined in the program RFA: DNA mutations, long-read sequence analysis, scRNA-Seq analysis, and spatial genomics data analysis (with connection to digital imaging analysis).