Establishing and benchmarking advanced methods to comprehensively characterize somatic genome variation in single human cells

摘要 理解体细胞基因组变异面临着独特的挑战，主要源于 在多细胞有机体中，大多数体细胞突变的个体稀有性。因此， 敏感度和准确度(由于需要区分体细胞变异和噪声)都变得 至关重要的是。散装DNA的分析，即使用超精密的方法，也只能确定 人类基因组的一部分。对单个细胞的分析，无论是通过克隆还是体外整体- 基因组扩增(WGA)，能够在理论上独立地发现细胞中的所有突变 它们的成批频率。然而，在体外扩增单细胞基因组仍然是一个 就扩增的准确性而言，这是一个重大挑战。新的PTA技术(主要 模板定向扩增)大大提高了扩增DNA的质量。然而， PTA产生的DNA片段相对较少，长度适中。这限制了 长读测序技术的应用。预计长读测序将是最多的 体细胞突变检测的综合方法。在家长奖励项目中，我们将首先， 从活体捐赠者的几个不同组织中克隆扩增的IPSC系，用于研究 利用非酶扩增基因组DNA进行所有类型的体细胞突变。第二，我们将 解决单细胞基因组分析的一个重大缺陷，即缺乏直接的 有关正在分析的细胞的确切类型的信息，或有关潜在功能的信息 该细胞突变的后果。为此，我们将对新的ResolveOme方法进行基准测试， 可以同时分析单个细胞的基因组和转录组。第三，我们将解决 对单细胞进行高通量分析以检测体细胞结构变异的挑战。 具体地说，我们将为SMAHT建立和基准Strand-seq方法，该方法允许 高通量检测和表征单细胞中的结构变异(SVS)。 总而言之，这将解决在分析正常组织中的体细胞突变时的3个关键需求： 全面的马赛克突变发现，对含有突变的细胞进行表型鉴定，并直接 评估突变的功能后果，以及准确和高通量检测 SVS. 在这个补充项目中，我们将从一个活体捐赠者那里培育出一个已经存在的成纤维细胞系， 以及从该成纤维细胞系衍生的现有的5个克隆扩增的IPSC系。我们 将把这些线路上的细胞分配给SMAT财团的GCC进行深度测序 为了对已经预先验证了体细胞变体的资源进行基准测试 在已知的组织等位基因频率上存在。

Abstract Understanding somatic genomic variation presents unique challenges, primarily stemming from the individual rarity of most somatic mutations across cells in a multicellular organism. Hence, both sensitivity and accuracy (due to the need to distinguish somatic variation from noise) become crucially important. The Analysis of bulk DNA, even with ultraprecise approaches, only ascertains a portion of the human genome. The analysis of single cells, either by cloning or in vitro whole- genome amplification (WGA), enables discovering theoretically all mutations in a cell independent of their frequency in bulk. However, amplifying single cell genomes in vitro represents still a significant challenge in terms of accuracy of amplification. The novel PTA technique (primary template directed amplification) offers substantially improved quality of amplified DNA. However, PTA produces a relatively small amount of DNA fragments of moderate length. This limits the application of long read sequencing. Long read sequencing is expected to be the most comprehensive approach to somatic mutation detection. In the parent award project, we will, first, produce clonally expanded iPSC lines from several different tissues of a live donor, to study somatic mutation of all types using non-enzymatically amplified genomic DNA. Second, we will address a significant shortcoming of the analysis of single cell genomes, which is the lack of direct information about the exact type of cell being analyzed, or about potential functional consequences of mutations in that cell. For that, we will benchmark the new ResolveOme method, that can analyze in parallel the genome and transcriptome of a single cell. Third, we will address the challenge of high-throughput analysis of single cells to detect somatic structural variants. Specifically, we will establish and benchmark for SMaHT the Strand-seq method that allows for high-throughput detection and characterization of structural variants (SVs) in single cells. Together, this will address 3 critical needs in the analysis of somatic mutations in normal tissues: comprehensive mosaic mutation discovery, phenotyping the cell harboring mutations and directly assessing functional consequences of mutations, and accurate and high-throughput detection of SVs. For this Supplement project, we will grow up an already-existing fibroblast line from a live donor, as well as five already existing clonally expanded iPSC lines derived from that fibroblast line. We will distribute cells from these lines to the GCCs of the SMaHT consortium, to be deep-sequenced for the purpose of benchmarking on a resource for which already pre-validated somatic variants at known tissue allele frequencies exist.