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

开发准确的高覆盖率和高通量单细胞 Duplex-seq 化学和多组学平台，用于同时分析单个人类细胞中的体细胞突变和转录组

• 批准号: 10662693
• 负责人: Chenghang Zong
• 金额: $ 39.78万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-04-15 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10662693
• 关键词: Aging; Bioinformatics; Biological Assay; Biological Process; Cells; Cellular Assay; Chemistry; DNA; DNA Damage; Data; Data Analyses; Detection; Development; Disease; Dissection; Encapsulated; Event; Frequencies; Future; Genome; Genomic DNA; Goals; Grant; Heterogeneity; High-Throughput Nucleotide Sequencing; Human; Individual; Informatics; Life; Malignant Neoplasms; Measurement; Methods; Microfluidics; Mosaicism; Mutation; Mutation Detection; Nerve Degeneration; Normal tissue morphology; Phase; Phenotype; Productivity; Publishing; RNA; Recovery; Resolution; Single Nucleotide Polymorphism; Somatic Mutation; Source; Standardization; System; Technology; Tissues; Trees; Validation; Variant; Work; age related; cost; de novo mutation; design; experience; genome sequencing; human disease; improved; innovation; medical specialties; method development; multiple omics; nano; programs; scale up; single-cell RNA sequencing; tool; transcriptome; transcriptomic profiling; whole genome

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.

概括 在这个 UG3/UH3 提案中，我们的目标是开发一种新的单细胞全基因组扩增 化学允许在单个细胞中高精度和高覆盖率地检测体细胞突变 细胞和结合高精度和高覆盖基因组的双组学检测 使用单细胞转录组分析 (UG3) 进行分析分析 在 UH3 中，我们的目标是扩大规模。 提高 scDuplex-seq 测定和相关双组学测定的通量 基于微微注射的液滴平台，并针对不同的组织类型验证该平台 SMaTH 计划将对其进行大规模的体细胞镶嵌分析。 单细胞分辨率下生命蓝图的异质性对于我们理解生命蓝图至关重要 许多基本的生物过程，例如衰老和癌症等人类疾病 因此，所提出的单细胞方法的成功开发是。 对于实现 SMaHT 计划设定的体细胞嵌合体分析目标非常重要 据我们所知，考虑到体细胞突变是最常发生的类型 随着方法开发的成功，我们可以确定整体的体细胞变异。 单细胞的突变负担及其之间的变异超出了表征的范围。 体细胞突变水平，我们还可以有效地为所有测序的构建谱系树 我们预计不同分支之间会存在表型差异。 谱系树的结构，对应于我们体内的不同克隆，正如最近在 基于区域解剖的研究。在识别出不同的分支/克隆后，我们可以 所提出的双组学测定将描述它们之间的表型差异。 就我们开发的主要策略而言，为这种表征提供了准确的工具。 准确的高覆盖率基因组分析方法，我们将应用专门的转座 对基因组 DNA 进行化学处理，产生片段大小非常均匀的双链 DNA， 我们的主要技术是在下游化学中最大限度地回收这些碎片。 扩大吞吐量的专长是微微注射液滴系统，它本质上允许 在液滴系统上实施复杂的化学反应。 Zong实验室和Weitz实验室之间的经验也被证明在以下方面是富有成效的： 液滴 scTotalRNA-seq 的开发 我们该提案的最终目标是产生一个 具有大量细胞（³1000）的谱系树，具有准确的体细胞特征 单细胞中的突变和转录组，从而提供了概念图的证明 SMaHT 计划的基因组表征中心未来进行大规模分析。