NOVEL METHODS FOR VAST INCREASE IN THROUGHPUT AND ACCURACY OF CIS-REG ANALYSES

描述（由申请人提供）：本申请涉及广泛的挑战领域（08）、基因组学和特定挑战主题08-HG-101“基因组序列中功能元件的高通量功能分析的技术和资源”。提出了一个为期两年的项目，以扩大，开发，延伸，并在示范的基础上应用一个全新的方法，高通量顺式调控分析。我们最近开发了这种新技术的初始组成部分，作为快速验证海胆胚胎基因调控网络模型的工具，并证明了其在促进发现和测量以前未知的顺式调控模块的定量活性方面的功效，其通量高达传统方法的100倍。该方法的基本原理是使用序列标记的“条形码化”载体，其可以在单个实验中大量地一起引入并且稍后去卷积。但是，还有许多额外的副产品和额外的发展需要付诸实践，挑战赠款计划提供了一个机会，安装一个“崩溃计划”，并把这些机会在不久的将来上线。此外，我们迄今为止开发的方法测量定量顺式调节输出，而不是空间活性。我们提出了额外的技术发展，以产生更高质量的空间表达数据比可通过任何其他手段和高通量的方法恢复大的顺式调控模块在任何给定的空间调控状态下运行。该提案的具体目标包括使序列标签方法适应NanoString技术，以允许同时评估> 100个不同顺式调节模块的活性;证明使用该方法同时获得大量顺式调节模块的时间输出曲线;通过使用NanoString测量，开发确定未知顺式调控模块的空间表达谱的非常高的准确性方法;并调整高通量技术以用于分离在生物体的给定时空域中操作的大量未知组的所有顺式调节模块的通用性。另外两点意见是重要的：第一，没有先验的理由说明为什么这些技术不能转移到任何其他利用直接DNA注射进行基因转移的系统中;第二，为了实现这些目标，我们必须建立一个新的研究小组。这将需要雇用更多的人员，在这方面，提案的科学和组织方面都与A.R.R.A.的目标协同增效。国家卫生研究院的倡议。这项工作是关于找到因果控制线，决定如何根据基因组调控系统中编码的指令执行基本生命过程。最有效的方法来解决复杂的疾病状态需要坚实的理解他们的控制电路。我们的实践必须超越努力改善影响，而不是改变原因。这项研究显示了因果基因组控制系统的结构和功能的发现的方式。

DESCRIPTION (provided by applicant): This Application addresses broad Challenge Area (08), Genomics, and Specific Challenge Topic 08-HG-101 "Technology and resources for high throughput functional analysis of functional elements in genomic sequences". A two year project is proposed to expand, to exploit, to extend, and to apply on a demonstration basis a completely novel approach to high throughput cis-regulatory analysis. We have very recently developed the initial component of this new technology as a tool for rapid validation of sea urchin embryo gene regulatory network models, and demonstrated its efficacy in facilitating the discovery and in measuring the quantitative activity of previously unknown cis-regulatory modules with a throughput of up to 100x that of traditional methods. The essential principle of this approach is use of sequence-tagged "barcoded" vectors which can be introduced together in large number in a single experiment and de-convolved later. But there remain many additional spinoffs and additional developments to be brought to practice, and the Challenge Grant program offers the opportunity to mount a "crash program" and bring these opportunities on line in the immediate future. In addition, the methods we have so far developed measure quantitative cis-regulatory output and not spatial activity. We propose additional technological developments to generate higher quality spatial expression data than obtainable by any other means and a high throughput method of recovering large sets of cis-regulatory modules operating in any given spatial regulatory state. The specific aims of this proposal include adapting the sequence tag method to NanoString technology to permit simultaneous assessment of activity of > 100 different cis-regulatory modules; demonstrate the use of this method to obtain temporal output profiles of large numbers of cis-regulatory modules simultaneously; develop a very high accuracy method of determining spatial expression profiles of unknown cis-regulatory modules by use of NanoString measurements; and tune for general use a high throughput technology for isolating all cis-regulatory modules of a large unknown set which operate in a given time-space domain of the organism. Two additional comments are important: first, there is no a priori reason why these technologies should not be transferrable to any other system in which gene transfer by direct DNA injection is utilized; and second, in order to accomplish these objectives we shall have to build a new research subgroup. This will require hiring additional personnel, and in this respect both the scientific and organizational aspects of the proposal synergize with the objectives of the A.R.R.A. NIH initiative. This work is about finding the causal lines of control that determine how fundamental life processes are executed according to the instructions encoded in the genomic regulatory system. The most powerful approach to general solutions to complex disease states requires solid understanding of their control circuitry. Our practice must get beyond struggling to ameliorate effects rather than altering causes. This research shows the way to discovery of structure and function in causal genomic control systems.