Hybrid approach for comprehensive mutation detection in a cell

摘要 最近的许多研究一致表明，可能没有两个细胞在人体内有 相同的基因组，这种现象被称为体细胞镶嵌。镶嵌现象可以使用多种方法进行研究， 但直接在细胞中研究突变，有望全面表征嵌合现象， 组织.通过克隆分析单细胞基因组依赖于细胞中的天然DNA复制机制，因此， 最大限度地减少克隆过程中DNA的错误;然而，克隆受到细胞增殖能力的限制。分析 通过全基因组扩增（WGA）的方法受到引入的误差和扩增的不均匀性的阻碍。 在这里，我们建议解决单细胞克隆和单细胞WGA的局限性，通过开发一个混合 该方法分两个阶段进行：1）将单个细胞有限培养成2-50个细胞的微型集落; 和2）微尺寸菌落的WGA以产生足够的DNA材料用于测序。一种优化的混合动力车 这种方法将能够在整个细胞周期中严格和无偏见地研究单细胞水平的体细胞镶嵌。 没有WGA伪影的人体。最后，为了保持组织细胞的异质性，并使生物库能够 组织适合开发的混合方法，我们将开发一个存储协议的组织保存 储存组织中细胞的增殖潜力。该项目的成功将使全面和 在SMaHT优先考虑的各种组织中的单个细胞中准确发现突变， 加深了我们对人类镶嵌性的理解。 2

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