High definition in vivo footprinting via single molecule sequencing

描述(申请人提供)：我们计划开发一种基于下一代单分子DNA测序技术的高通量、高分辨率活体足迹方法。单分子测序将取代传统的方法来检测三种修饰剂：DMS(硫酸二甲酯)、DNase I和紫外光诱导的切割。通过对标准连接介导的聚合酶链式反应(LM-PCR)的简单修改，蛋白质结合位点的精确序列可以通过对短切割签名标签进行测序并简单地计算标签在指定的‘足迹窗口’内的每个核苷酸位置终止的次数来确定。这种计数方法将把传统足迹方法繁琐的“带强度”分析转变为一种绝对的、基于频率的方法，可以很容易地通过自动分析管道进行分析。该项目从该技术的开发和示范及其大规模使用开始。首先，将开发最佳条件并将其应用于以多路复用格式生成先前已印有足迹的“控制”片段(DMS、DNase I和UV)的切割图。建立了优化的参数后，该方法将用于推测顺式调控元件的从头足迹，如DNase I超敏反应所示。在这一目标中，我们还将确定HD(高清晰度)标签足迹分析的灵敏度和特异性，以及评估该系统的生物和技术重复性。&gt；100个标签生成的足迹每年都将通过经典的足迹技术进行验证，以确保数据的准确性。最后，我们将确定HD标签足迹系统的可扩展性，并将其大规模应用于ENCODE区域内假定的功能元件的分析。从四种细胞类型确定的DNase I超敏感位点将被打印出来，并与现有的ENCODE数据类型进行比较。一种能够产生高分辨率DNase I、DMS和UV足迹的高通量方法将为鉴定任何真核基因组中新的顺式调控元件中的蛋白质相互作用序列提供强有力的工具。高分辨率标签足迹将通过补充现有的分析并创建大量精确的基于序列的数据来促进ENCODE项目的目标，这些数据可用于进一步注释基因组中的功能元件。

DESCRIPTION (provided by applicant): We plan to develop a high-throughput, high-resolution in vivo footprinting method based on next generation single molecule DNA sequencing technology. Single molecule sequencing will be used in place of conventional methods to detect cleavage induced by three modifying agents: DMS (dimethyl sulfate), DNase I and UV light. With a simple modification to standard ligation-mediated PCR (LM-PCR), the precise sequence of protein binding sites can be determined by sequencing short cleavage signature tags and simply counting the number of times a tag terminates at each nucleotide position within a designated 'footprint window'. This counting method will convert the cumbersome, 'band intensity' analysis of classical footprinting methods to an absolute, frequency-based approach that can be readily analyzed by an automated analysis pipeline. The project commences with the development and demonstration of the technology and its large-scale use. First, optimal conditions will be developed and applied to generate cleavage maps of previously footprinted 'control' segments (DMS, DNase I and UV) in a multiplexed format. With optimized parameters established, the assay will be used for the de novo footprinting of putative cis regulatory elements, as indicated by DNase I hypersensitivity. Within this aim we will also determine the sensitivity and specificity of the HD (High Definition) tag footprinting assay as well as assess the biological and technical reproducibility of the system. > 100 tag-generated footprints will be validated each year by classical footprinting techniques to ensure accuracy of the data. Finally, we will determine the scalability of the HD tag footprinting system and apply it on a large scale to the analysis of putative functional elements within the ENCODE regions. DNase I hypersensitive sites identified from four cell types will be footprinted and data compared to existing ENCODE data types. A high-throughput method capable of generating high resolution DNase I, DMS and UV footprints will provide a powerful tool for identifying the protein interacting sequences within novel cis-regulatory elements of any eukaryotic genome. High-resolution tag footprinting will contribute to the goals of the ENCODE project by complementing existing assays and creating a large volume of precise sequence-based data that can be used to further annotate functional elements in the genome.