Hybrid approach for comprehensive mutation detection in a cell

摘要 最近的大量研究一致表明，人体内可能没有两个细胞具有 同样的基因组，这种现象被称为体细胞嵌合体。可以使用各种方法来研究马赛克， 但是对直接在细胞中的突变的研究保证了对任何 组织。通过克隆来分析单细胞基因组依赖于细胞中的自然DNA复制机制，因此， 最大限度地减少克隆过程中的DNA错误；然而，克隆受到细胞增殖能力的限制。分析 全基因组扩增(WGA)过程中引入的误差和扩增的不一致性阻碍了WGA的实现。 在这里，我们建议通过开发杂交种来解决单细胞克隆和单细胞WGA的局限性 该方法分两个阶段进行：1)将单个细胞的培养限制在2-50个细胞的微型集落； 2)微型菌落的WGA，以产生足够的DNA材料用于测序。一种优化的混合动力车 这一方法将使在整个 没有WGA人工制品的人体。最后，为了保持组织细胞的异质性并使生物库 适用于开发的混合方法的组织，我们将开发一种组织存储方案来保存 储存组织中细胞的增殖能力。该项目的成功将使全面和 在SMAT及其他优先考虑的各种组织中准确发现单个细胞的突变， 加深了我们对人类马赛克的理解。 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