Project Summary Somatic mosaicism results from the accumulation of mutations over time, leading to a unique genotype in every cell. These mutations can be as small as a single base pair or as large as an entire chromosome – affecting hundreds or even thousands of genes. Mosaic chromosomal alterations (mCA) are one type of somatic mosaicism characterized by large chromosomal gains or losses and are associated with aging, heart disease, kidney disease and cancer. The role of mCA has been best characterized in blood, but recently we used single- cell sequencing to show that mCA are also common in the kidney and are associated with disease progression. Kidney and blood are hotspots for mCA, but these data raise the possibility that mCA are present in other organs where they exert tissue-specific effects. Existing algorithms for mCA detection are tailored to cancer and rely on the assumption that cancer cells are clonal and carry the same variants. This is a significant limitation because mCA in non-neoplastic tissue are not clonal and may only be present in a small fraction of cells. Moreover, mCA that do not affect copy number or transcript abundance and do not reside in open chromatin regions are all undetectable with current methods. This proposal aims to develop methods to improve detection of mCA that will enable novel lines of inquiry in kidney, blood and other non-neoplastic tissues. Our single-cell methods will be complemented by algorithms to localize mCA in spatial transcriptomics datasets, which may provide insight into how mCA affect disease progression via interaction with neighboring cells. In addition, we will design high- throughput assays that can be used to evaluate mCA burden at low cost in a wide variety of sample types. All methods will be released as open-source protocols and software packages that researchers can tailor to their tissue of interest and may lead to novel therapies that aim to eliminate cells with mCA.