A modular, customizable sequencing system for simultaneous genotyping and transcript analysis in single cells

模块化、可定制的测序系统，用于在单细胞中同时进行基因分型和转录本分析

• 批准号: 9901478
• 负责人: Manuel Garber
• 金额: $ 18.22万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-04-01 至 2022-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9901478
• 关键词: Address; Affect; Alleles; Bar Codes; Biological Assay; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; Custom; DNA; DNA-Directed DNA Polymerase; Disease; Elements; Encapsulated; Enhancers; Ensure; Female; Gene Expression; Genes; Genetic; Genetic Diseases; Genetic Transcription; Genetic Variation; Genome; Genomic DNA; Genomics; Genotype; Gold; Heterogeneity; Hour; Human Genetics; Hybrid Cells; Hybrids; Individual; Malignant Neoplasms; Measures; Methods; Microfluidics; Mutagenesis; Mutation; Nature; Oligonucleotides; Output; Population; Population Heterogeneity; Population Sizes; Process; Protocols documentation; RNA; Reaction; Regulatory Element; Resolution; Reverse Transcription; Screening Result; Sensitivity and Specificity; Somatic Mutation; Specificity; Surveys; System; Technology; Testing; Tissues; Transcript; Untranslated RNA; X Inactivation; base; biological systems; cell type; cost; design; falls; flexibility; human disease; innovation; innovative technologies; interest; manufacturing process; novel; sequencing platform; single cell sequencing; single cell technology; single molecule; single-cell RNA sequencing; targeted sequencing; transcriptome; transcriptome sequencing; tumor

Project Summary Single cell sequencing has revolutionized the way in which we define cell types and understand tissues, and has tremendous potential for analyzing the heterogeneity of complex tumors or large perturbation screens. At present, however, this powerful technology has serious limitations, particularly in what cellular information can be detected and analyzed. Current high-throughput microfluidics single cell RNA sequencing (scRNA-seq) methods can conveniently process tens of thousands of cells but only capture the extreme 3’ ends of the most abundant transcripts from each cell. The low sensitivity and partial transcript coverage hampers these methods’ abilities to detect allele-specific or subtle perturbation effects, while also dramatically increasing the per-cell sequencing cost. Moreover, no existing single-cell sequencing method can read DNA genotype and RNA expression from the same cell, which is crucial to studying non-coding regulatory elements and somatic rearrangements -- where the most genetic variation associated with cancer and human disease resides. Here we propose to overcome these limitations by developing a flexible high throughput single-cell sequencing system that (1) Can target many different regulatory DNA elements and ​transcripts simultaneously in the same cell​, (2) Is sensitive enough to measure subtle, allele-specific effects, (3) measures the effects of different mutations across tens-of-thousands of cells in a single assay, and (4) is easily and rapidly adaptable for application to any biological system with a heterogeneous cell population. We iteratively develop this technology, ensuring that each step independently creates new capabilities that address current scRNA-Seq limitations and enables allele specific expression analysis and perturbation screens of non-coding elements. First​, we will modify the the inDrop bead manufacturing process to make it flexible and rapidly customizable so that one large batch of universal barcoded beads can be conveniently adapted to target many specific transcript pools, SNP-containing portions of transcripts, and even genomic DNA, in just 8 hours. Second​, we test the sensitivity and allele-specificity of our new method in a predictably heterogeneous system: random X inactivation in hybrid female (XX) cells. Using single-molecule RNA-FISH as a gold standard, we will measure the sensitivity, specificity, and efficiency of our targeted scRNA-Seq approach. Third​, we enable simultaneous DNA genotyping and transcript quantification by adapting our custom beads and reaction conditions for isothermal amplification of genomic DNA loci, simultaneous with RT of targeted transcripts in the same cells. Fourth​, we will combine our approaches above in a proof-of-principle application to characterize enhancer function using a CRISPR mutagenesis screen. CRISPR mutagenesis randomly creates different alleles in each cell. We then use targeted sequencing of neighboring genes and DNA genotyping to evaluate the effect of each allele on its target(s) in ​cis​.

项目概要 单细胞测序彻底改变了我们定义细胞类型和了解组织的方式，并已 分析复杂肿瘤或大扰动屏幕的异质性的巨大潜力。在 然而，目前这种强大的技术存在严重的局限性，特别是在细胞信息方面 可以被检测和分析。当前高通量微流控单细胞 RNA 测序 (scRNA-seq) 这些方法可以方便地处理数以万计的细胞，但只能捕获大多数细胞的最末端 3' 端。 每个细胞都有丰富的转录本。低敏感性和部分转录本覆盖阻碍了这些 方法检测等位基因特异性或微妙的扰动效应的能力，同时还显着提高 每个细胞的测序成本。此外，现有的单细胞测序方法还没有能够读取DNA基因型和 来自同一细胞的 RNA 表达，这对于研究非编码调控元件和体细胞至关重要 重排——与癌症和人类疾病相关的遗传变异最多的地方。 在这里，我们建议通过开发灵活的高通量单细胞测序来克服这些限制 系统 (1) 可以同时靶向许多不同的调控 DNA 元件和转录本 相同细胞​，(2) 足够灵敏，可以测量微妙的等位基因特异性效应，(3) 测量不同细胞的影响 在一次测定中跨越数万个细胞的突变，并且（4）可以轻松快速地适应 适用于任何具有异质细胞群的生物系统。我们迭代开发这个 技术，确保每个步骤独立创建解决当前 scRNA-Seq 问题的新功能 限制并实现等位基因特异性表达分析和非编码元件的扰动筛选。 首先，我们将修改 inDrop 珠子制造工艺，使其灵活且可快速定制 这样一大批通用条形码珠子就可以方便地适应许多特定的目标 只需 8 小时，即可获得转录本池、转录本中包含 SNP 的部分，甚至基因组 DNA。 其次，我们在可预测的异质系统中测试新方法的敏感性和等位基因特异性： 杂交雌性 (XX) 细胞中的随机 X 失活。使用单分子 RNA-FISH 作为金标准，我们 将测量我们的靶向 scRNA-Seq 方法的灵敏度、特异性和效率。 第三，我们通过调整我们的定制微珠实现同步 DNA 基因分型和转录本定量 以及基因组 DNA 位点等温扩增的反应条件，同时进行目标 RT 同一细胞中的转录本。 第四，我们将在原理验证应用中结合上述方法来表征增强器 使用 CRISPR 诱变筛选功能。 CRISPR 诱变随机在每个基因中产生不同的等位基因 细胞。然后，我们使用邻近基因的靶向测序和 DNA 基因分型来评估每个基因的效果 顺式目标上的等位基因。