Developing an ultra-high throughput droplet microfluidic workflow for genetic circuit characterization

项目总结 现有的单细胞测序技术对独特的基因组提供了前所未有的理解 以及异质生物样本背后的转录差异。这些差异是 了解临床标本中的疾病病理和基础生物学的基本机制 申请。最近的注意力集中在多组体技术的发展上，这种技术可以更好地 捕获配对数据集中的差异，如基因组和转录组或转录组，并 表观基因组。然而，目前还没有一种单细胞技术可以用 细胞的相应表型行为。 本项目的目的是开发一种新的测序平台来解决这一技术差距。至 演示这个平台的实用性，然后我将应用它来快速表征可调基因振荡器。 为了实现这一目标，我提出了以下两个具体目标。在目标1中，我将开发FAB-SEQ (荧光注释条形码和测序)。我将首先演示一种新型的双重条形码 一种联合传递光学和DNA条形码以在显微镜图像之间创建单细胞映射的方法 数据和序列数据。我还将展示FAB-SEQ可用于进行靶向测序 一种任意的表型。然后，我将利用额外注入的DNA条形码来增强总条形码 Fab-Seq空间在目标2中，我将演示FAB-SEQ可用于快速表征可调参数 基因振荡器。首先，我将演示FAB-SEQ可以从电路库中检测到振荡表型 包含振荡和非振荡遗传电路的。然后我将展示，当振动行为 电路受到扰动时，FAB-SEQ可以将单个振荡动力学映射到相应的单个单元 转录组。 该项目的长期目标是开发一种可以映射单细胞显微镜数据的平台技术 到超高吞吐量的单细胞测序数据。我设想Fab-seq将成为一个变革性的工具 解决基因组学领域前沿的问题。

PROJECT SUMMARY Existing single cell sequencing technologies provide an unprecedented understanding of the unique genomic and transcriptomic differences that underlie heterogeneous biological samples. These differences are key to understand disease pathologies in clinical samples and fundamental mechanisms in basic biological applications. Recent attention has been directed towards the development of multiomic technologies that better capture the differences in paired datasets such as genome and transcriptome or transcriptome and epigenome. Currently however, there is no single cell technology that can profile sequence information with the corresponding phenotypic behavior of the cell. The purpose of this project is to develop a novel sequencing platform to address this technology gap. To demonstrate the utility of this platform, I will then apply it to rapidly characterize a tunable genetic oscillator. To accomplish this goal, I propose the following 2 specific aims. In Aim 1, I will develop FAB-seq (Fluorescence Annotated Barcoding and Sequencing). I will first demonstrate a novel dual barcoding approach that co-delivers optical and DNA barcodes to create single cell maps between microscopy image data and sequence data. I will also show that FAB-seq can be used to perform targeted sequencing according to an arbitrary phenotype. Then, I will leverage additional injected DNA barcodes to enhance the total barcode space of FAB-seq. In Aim 2, I will then demonstrate that FAB-seq can be used to rapidly characterize a tunable genetic oscillator. First, I will demonstrate that FAB-seq can detect oscillatory phenotypes from a circuit library containing oscillatory and non-oscillatory genetic circuits. Then I will show that when the oscillatory behavior of the circuit is perturbed, FAB-seq can map individual oscillation dynamics to the corresponding single cell transcriptome. The long-term goal of this project is to develop a platform technology that can map single cell microscopy data to single cell sequencing data at ultra-high throughput. I envision FAB-seq to be a transformative tool in addressing questions at the frontier of the genomics field.