Project Summary - Genome Characterization Unit (GCU) The GCU will provide comprehensive, end-to-end CLIA-compliant genomic testing and cutting-edge research- level analysis of patients with MM, CRC, and CHOL who are engaged by the PEU. This testing will be conducted on diagnostic tumor samples and matched normal tissue from 300 patients for each of the three cancer types during the WU PE-CGS research program. Additional follow-up samples from 150 of these patients will also be analyzed during disease progression. Sample processing by the GCU will include nucleic acid extraction and QC. Diagnostic samples will be analyzed using 250X tumor/normal exome sequencing (WES) with both somatic and germline analysis for gene-level single nucleotide variants (SNV and insertion/deletions (indels). Tumor-only WGS (60X) will be performed to detect tumor-associated structural mutations, and tumor RNA-seq will support, confirm, and extend findings from the DNA-based assays. Tumor tissue and/or cell-free DNA will also be analyzed with targeted deep sequencing (>10,000X) using unique molecular indexes (UMI) for sensitive detection and monitoring of tumor-associated mutations. All of these assays will be performed using CLIA-compliant procedures with integrated quality management practices. The GCU will also conduct research-level scRNA-Seq, proteomics, and cellular imaging studies on selected samples to enhance our understanding of these tumor types. Genomic assays will proceed according to a planned schedule for year-by-year combinations of diagnostic specimens and follow-up collections, with small numbers of candidates selected for research studies. Results from these assays will be returned to participants using a tiered reporting system that will depend on participant preference. Tier 1 results will highlight findings with established clinical relevance obtained from CLIA sequencing of individual participants, including pathogenic, tumor-associated somatic drivers and inherited mutations that are clinically actionable according to published guidelines and that will be reported using established categorization for somatic drivers and pathogenic germline variants, their clinical implications, and possible actions. Participants can also elect to receive Tier 2 results, which will be comprised of additional mutations from the same CLIA-compliant data that are identified with advanced methods and are predicted to be clinically relevant via functional annotation, as well as results from targeted sequencing of follow-up samples for monitoring tumor evolution over time. Research-level Tier 3 molecular studies may also be provided to participants as aggregate, deidentified reports that can be used to enhance and extend interpretations of their individualized CLIA results. These results will also be securely uploaded to the NCI Genomic Data Commons (GDC) for use by the cancer biology community. All GCU activities will be coordinated with the PEU and EOU to ensure clarity and consistency across the WU PE-CGS program.