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

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， 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，这种强大的技术有严重的局限性，特别是在什么样的蜂窝信息， 可以检测和分析。当前高通量微流体单细胞RNA测序（scRNA-seq） 方法可以方便地处理数万个细胞，但仅捕获大多数细胞的末端3'端。 每个细胞都有大量的转录本低灵敏度和部分转录覆盖阻碍了这些 方法检测等位基因特异性或细微扰动效应的能力，同时也显著提高了 每细胞测序成本。此外，没有现有的单细胞测序方法可以读取DNA基因型， 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生物学至关重要。 基因重排--与癌症和人类疾病相关的最多的基因变异所在。 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）足够灵敏以测量细微的等位基因特异性效应，（3）测量不同细胞的效应， 在一个单一的测定中，在成千上万的细胞中检测突变，以及（4）容易且快速地适用于 本发明还适用于具有异质细胞群的任何生物系统。我们反复开发这个 技术，确保每一步都独立地创造新的能力，解决当前的scRNA-Seq 限制，并且能够进行等位基因特异性表达分析和非编码元件的干扰筛选。 首先，我们将修改inDrop珠的制造过程，使其具有灵活性和可快速定制性 使得一大批通用条形码化珠可以方便地适用于靶向许多特定的 在短短8小时内，就可以完成转录物池、含SNP的转录物部分，甚至基因组DNA。 其次，我们在可预测的异质系统中测试了我们的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-FISH作为金标准，我们 将测量我们靶向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珠粒， 以及用于基因组DNA基因座的等温扩增的反应条件，与靶向的 在相同的细胞中。 第四，我们将联合收割机结合我们的方法在一个证明的原则应用程序，以表征增强 使用CRISPR诱变筛选来鉴定其功能。CRISPR诱变在每个基因组中随机产生不同的等位基因。 cell.然后，我们使用相邻基因的靶向测序和DNA基因分型来评估每种方法的效果。 等位基因在其靶标上呈顺式。

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​.