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​.

项目摘要 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，并具有 分析复杂肿瘤或大扰动筛选的异质性的巨大潜力。在 但是，目前，这项强大的技术有严重的局限性，尤其是在哪些蜂窝信息中 可以检测和分析。当前的高通量微流体单细胞RNA测序（SCRNA-SEQ） 方法可以方便地处理成千上万个单元，但只捕获了最多的极端3'末端 来自每个单元的最富有的转录本。低灵敏度和部分成绩单覆盖范围阻碍了这些 方法检测特定或微妙的扰动效果的能力，同时也大大增加了 每个细胞测序成本。此外，没有现有的单细胞测序方法可以读取DNA基因型和 来自同一细胞的RNA表达，这对于研究非编码调节元件和体细胞至关重要 重排 - 与癌症和人类疾病有关的最大遗传变异。 在这里，我们建议通过开发灵活的高吞吐量单细胞测序来克服这些限制 （1）可以针对许多不同的调节DNA元素和转录本的系统 相同的细胞，（2）足够敏感，可以测量微妙的等位基因特异性效应，（3）测量不同的影响。 单个测定中数以万计的细胞之间的突变，（4）容易适应于 应用于具有异质细胞群体的任何生物系统。我们迭代地开发这个 技术，确保每个步骤都独立创建新的功能来解决当前的scrna-seq 限制并使非编码元素的等位基因表达分析和扰动筛选。 首先，我们将修改Indrop珠子制造过程，以使其灵活且可快速自定义 因此，可以方便地适应一大批通用条形码珠，以针对许多特定 在短短8个小时内，转录本库，含SNP的转录本甚至基因组DNA。 其次，我们在一个可以预见的异质系统中测试了新方法的灵敏度和等位基因特异性： 杂交雌性（XX）细胞中的随机X失活。使用单分子RNA鱼作为黄金标准，我们 将衡量我们目标SCRNA-SEQ方法的灵敏度，特异性和效率。 第三，我们通过适应我们的自定义珠来启用同时进行DNA基因分型和转录本定量 和反应条件，用于基因组DNA基因座的等热扩增，同时与靶向的RT 同一单元格中的转录本。 第四，我们将在原理证明应用中结合上述方法以表征增强器 使用CRISPR诱变屏幕的功能。 CRISPR诱变会随机创建不同的等位基因 细胞。然后，我们使用邻近基因和DNA基因分型的靶向测序来评估每个基因的效果 在其目标中的靶标上等位基因。