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

建立先进方法并对其进行基准测试，以全面表征单个人类细胞的体细胞基因组变异

• 批准号: 10662975
• 负责人: Alexander Eckehart Urban
• 金额: $ 39.43万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-04-19 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10662975
• 关键词: Autopsy; Benchmarking; Blood; Blood Cells; Blood Vessels; Bone Marrow; Brain; Cell Line; Cells; Characteristics; Clonal Expansion; Clone Cells; Cloning; DNA; DNA Insertion Elements; DNA amplification; Data; Detection; Disadvantaged; Endothelial Cells; Epithelial Cells; Failure; Fibroblasts; Freezing; Frequencies; Genes; Genome; Genomic DNA; Genomic Segment; Haplotypes; Human; Human Genome; Human body; In Vitro; Individual; Length; Methods; Microglia; Mosaicism; Mutation; Mutation Detection; Neuroglia; Neurons; Noise; Normal tissue morphology; Nucleosomes; Organism; Phase; Phenotype; Production; Resolution; Resources; Sampling; Skin; Somatic Mutation; Techniques; Technology; Testing; Tissue Procurements; Tissues; Urine; Variant; cell type; comparative; genome analysis; genome-wide; genomic variation; high throughput analysis; improved; induced pluripotent stem cell; insertion/deletion mutation; method development; mosaic variant; novel; novel strategies; single cell analysis; stem; telomere; tool; transcriptome; variant detection; whole genome

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 proposed project, we will, first, perform long-read sequencing in single cells cloned via the production of iPSC lines to study somatic mutation of all types using non-enzymatically amplified genomic DNA, from telomere to telomere, and generate a gold-standard benchmarking resource for methods development. 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, an expansion of PTA, 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. Another important aspect of the project will be comprehensive comparative analyses of detected somatic variants across all Aims.

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。在拟议的项目中，我们将首先， 对通过生产IPSC系克隆的单细胞进行长读测序研究 使用非酶扩增基因组DNA的所有类型的体细胞突变，从端粒到 端粒，并为方法开发生成黄金标准基准资源。 第二，我们将解决单细胞基因组分析的一个重大缺陷，即 是缺乏关于被分析的细胞的确切类型的直接信息，或者关于潜在的 该细胞突变的功能后果。为此，我们将对新的 ResolveOme方法是PTA的一种扩展，可以并行分析基因组和 单个细胞的转录组。第三，我们将应对高通量分析的挑战 用于检测体细胞结构变异的单个细胞。具体地说，我们将建立和基准 SMAHTSTRAND-SEQ方法，允许高通量检测和表征 单细胞中的结构变体(SVs)。总而言之，这将解决分析中的3个关键需求 正常组织中的体细胞突变：全面的马赛克突变发现，表型 含有突变并直接评估突变的功能后果的细胞，以及 准确、高通量的SVS检测。该项目的另一个重要方面将是 对检测到的所有目标的体细胞变异进行全面的比较分析。