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 在此UG3/UH3提案中，我们旨在开发新的单细胞全基因组扩增 化学允许高准确性和高覆盖率检测单个体细胞突变 细胞和双词分析结合了高临界性和高覆盖基因组 使用单细胞转录组测定法（UG3）进行分析测定。在UH3中，我们的目标是扩展 在scduplex-seq分析的吞吐量和相关的双向分析上 基于PicoInfoctive的液滴平台，并验证此平台的不同组织类型 大规模的Smath计划将通过Smath计划为躯体镶嵌介绍。理解 单细胞解决中生命蓝图的异质性对于我们对我们的理解至关重要 许多基本的生物学过程，例如衰老和人类疾病，例如癌症和 神经变性。因此，提出的单细胞方法的成功开发是 对于实现SMAHT计划设定的躯体镶嵌的目标很重要 据我们所知，考虑到躯体突变是最常见的类型 躯体变体。通过成功的方法开发，我们可以确定整体体细胞 单个细胞中的突变烧伤及其之间的变化。超越表征 体细胞突变的水平，我们还可以有效地为所有测序的谱系树构造一棵谱系树 单细胞。我们预计不同分支之间的表型差异 谱系树的相对应与我们体内不同的克隆相对应 基于区域解剖的研究。确定不同的分支/克隆后，我们可以 表征它们之间的表型差异。拟议的双词分析将 提供此特征的确切工具。就我们开发一个主要战略而言 准确的高覆盖基因组分析方法，我们将应用专门的换位 化学对基因组DNA，从而导致双链-DNA具有非常均匀的片段大小， 最大化这些片段在下游化学中的恢复。我们的主要技术 扩大吞吐量的专业是picoInfoctect液滴系统，本质上允许 在液滴系统上实施复杂的化学反应。协作 Zong Lab和Weitz Lab之间的经验也已被证明在 液滴sctotalrna-seq的开发。该提议的最终目标是生产 谱系树，具有大量的单元格（“ 1000”），具有躯体的两个精确特征 突变和单细胞中的转录组，因此提供了概念证明图片的证明 基因组表征中心的未来大规模分析中心。