Develop accurate high-coverage and high-throughput single-cell Duplex-seq chemistry and multi-omics platforms for simultaneous profiling of somatic mutation and the transcriptome in single human cells

总结 在这个UG 3/UH 3提案中，我们的目标是开发一种新的单细胞全基因组扩增技术， 化学，允许高准确度和高覆盖率检测单个细胞中的体细胞突变， 细胞和双组学分析，结合了高准确性和高覆盖率的基因组 用单细胞转录组测定（UG 3）进行谱分析测定。在UH 3中，我们的目标是 提高scDuplex-seq检测和相关双组学检测在 基于皮可注射的液滴平台，并验证该平台用于不同的组织类型， 将通过SMaTH程序进行大规模的体细胞镶嵌分析。理解 单细胞分辨率下生命蓝图的异质性对于我们理解 许多基本的生物过程，如衰老和人类疾病，如癌症， 神经变性因此，所提出的单细胞方法的成功开发是 这对于实现SMaHT计划设定的体细胞镶嵌性分析目标非常重要 考虑到体细胞突变，据我们所知，是最常见的类型， 体细胞变异有了成功的方法开发，我们可以确定整体体细胞 单个细胞中的突变负荷以及它们之间的变化。除了描述 水平的体细胞突变，我们也可以有效地构建一个谱系树，为所有测序 单细胞而且我们预计不同的分支之间会有表型差异 的谱系树，这对应于不同的克隆在我们的身体，最近观察到的 区域解剖研究。在识别不同的分支/克隆后，我们可以 描述它们之间的表型差异。所提出的双组学测定将 提供了准确的工具来描述这一特征。就我们发展一个 准确的高覆盖率基因组分析方法，我们将应用专门的转座 化学作用于基因组DNA，这导致了具有非常均匀的片段大小的cDNA， 最大化这些片段在下游化学中的回收。我们的主要技术 在按比例增加生产量方面的专长是皮可注射液滴系统， 在液滴系统上实施复杂的化学过程。合作 Zong实验室和Weitz实验室之间的经验也被证明是富有成效的， 液滴scTotalRNA-seq的开发。我们这项建议的最终目的是要产生一个 具有大量细胞（<$1000）的谱系树，具有体细胞的准确表征 突变和单细胞中的转录组，从而为 SMaHT计划的基因组表征中心未来的大规模分析。

SUMMARY Here in this UG3/UH3 proposal, we aim to develop a new single-cell whole-genome amplification chemistry that allows high-accuracy and high-coverage detection of somatic mutations in single cells and the dual-omics assay that combines the high-accuracy and high-coverage genome profiling assay with the single-cell transcriptome assay (UG3). And in the UH3, we aim to scale up the throughput of the scDuplex-seq assay and the related dual-omics assay on the picoinjection-based droplet platform and validate this platform for different tissue types that are going to be profiled for somatic mosaicism by SMaTH program at the large scale. Understanding the heterogeneity of the blueprint of life at single-cell resolution is critical for our understanding of many fundamental biological processes such as aging and human diseases such as cancer and neurodegeneration. Hence, the successful development of the proposed single-cell method is important for reaching the goals of profiling somatic mosaicism set by the SMaHT program considering that somatic mutations, to our knowledge, are the most frequently occurred type of somatic variants. With the successful method development, we can determine the overall somatic mutation burdens in single cells and the variations among them. Going beyond characterizing the levels of somatic mutations, we can also effectively construct a lineage tree for all the sequenced single cells. And we expect that there will be phenotypic differences between different branches of the lineage tree, which correspond to different clones in our body, as recently observed in the regional dissection-based studies. Upon identifying the different branches/clones, we can characterize those phenotypic differences between them. The proposed dual-omics assay will provide the exact tool for this characterization. In terms of our major strategy in developing an accurate high-coverage genome profiling method, we will apply specialized transposition chemistry to genomic DNA, which results in duplex-DNA with very uniform fragment size, maximizing the recovery of these fragments in the downstream chemistry. Our major technical specialty in scaling up the throughput is the picoinjection droplet system that essentially allows the implementation of complicated chemistry onto the droplet system. The collaborative experience between Zong lab and Weitz lab has also been proven to be productive in the development of the droplet scTotalRNA-seq. Our ultimate goal of this proposal is to produce a lineage tree with a large number of cells (³1000) with both accurate characterizations of somatic mutations and the transcriptome in single cells, hence providing the proof of concept picture for future large-scale profiling by Genome Characterization Centers of the SMaHT program.