Abstract Numerous recent studies have consistently shown that likely no two cells in the human body have the same genomes, a phenomenon called somatic mosaicism. Mosaicism can be studied using various approaches, but the study of mutations directly in the cell promises a comprehensive characterization of mosaicism in any tissue. Analysis of single cell genome by cloning relies on natural DNA replication machinery in cells and, thus, minimizes errors in DNA during cloning; however, cloning is limited by the ability of cells to proliferate. Analysis by whole genome amplification (WGA) is hampered by introduced errors and non-uniformity of amplification. Here we propose to address the limitations of single cell cloning and single cell WGA by developing a hybrid approach that proceeds in two stages: 1) limited culturing of single cells to a micro-sized colony of 2-50 cells; and 2) WGA of the micro-size colonies to yield enough DNA material for sequencing. An optimized hybrid approach will enable rigorously and unbiasedly studying somatic mosaic at a single cell level throughout the human body without WGA artifacts. Finally, to preserve tissue cell heterogeneity and enable biobanking of tissues amenable to the developed hybrid approach, we will develop a storing protocol for tissues to preserve proliferative potential of cells in the stored tissues. Success of the project would enable comprehensive and accurate discovery of mutations in a single cell in a variety of tissues prioritized by SMaHT and beyond, deepening our understanding of the mosaicism of humans. 2

Narrative Numerous recent studies have consistently shown that likely no two cells in the human body have the same genomes, a phenomenon called somatic mosaicism. Mosaicism can be studied using various approaches, but the study of mutations directly in the cell promises a comprehensive characterization of mosaicism in any tissue. However, all existing methods have significant limitations for studying the genome in a cell. Here we propose to develop and test a hybrid approach, which allows comprehensive and accurate analysis of the cell’s genomes in various tissues of the human body.