Ultrafast SBS Method for Large-Scale Human Resequencing

描述(由申请人提供)：识别和了解单核苷酸多态(SNPs)的作用将导致对遗传性疾病状态的准确诊断，风险因素的确定，以及患者代谢特征的表征。这种技术有望带来预防治疗，以延缓疾病的发生或发展，并提供最安全、最有效的药物处方。然而，目前的DNA测序技术对于这些任务来说太慢了，也太昂贵了。 在这里，我们建议开发一种超快DNA测序系统，其特点是在高密度寡核苷酸阵列上进行合成测序(SBS)，每个阵列具有大约一百万个引物特征。参与该项目的协作团队负责一些最早发表的关于SBS的工作，并认识到任何基于这种方法的方法在取得实际进展之前必须解决的根本挑战。也就是说，鉴定适当修饰的核苷三磷酸盐的组合，这些组合将被适当突变的DNA复制酶有效地和高保真地接受。因此，这项建议具有强大的合成化学成分，包括两个实验室，专注于制备带有荧光的、不稳定的3‘-保护基团的核苷三磷酸盐。它还描述了产生相对大的突变聚合酶文库的分子生物学。尽管产生的修饰酶的数量很高，但突变将集中在关键的结构区域，以最大限度地增加找到合适系统的机会。这种分子生物学成分与组合筛选相结合，以快速鉴定合适的酶/修饰的dNTP对。一旦确定了合适的组合，则将使用高密度阵列来实施SBS方法，该高密度阵列唯一地将寡核苷酸定向到所需的5‘->3’方向。这项核心技术符合更广泛的全面研究计划，包括用于样本处理和DNA传输到SBS系统的微流体、通过我们专有的脉冲-多线激发(PME)系统进行的荧光成像、确定最佳平铺路径的计算方法和整个染色体上寡核苷酸的热力学性质，以及处理和存储产生的数据的信息学。总的目标是，到第三年年底，完成3号、12号和X号染色体的测序，覆盖大约0.5亿个碱基，为全基因组测序奠定基础。

DESCRIPTION (provided by applicant): Identifying and understanding roles of single nucleotide polymorphisms (SNPs) will lead to accurate diagnosis of inherited disease states, determination of risk factors, and characterization of patients' metabolic profiles. Such technology promises to lead to prophylactic treatments to delay the onset or progression of disease, and prescriptions of the safest and most efficacious medications. Current DNA sequencing technology, however, is too slow and expensive for these tasks. Here, we propose to develop an ultrafast DNA sequencing system featuring sequencing-by-synthesis (SBS) on high-density oligonucleotide arrays, each with approximately one million primer features. The collaborative team involved in this project was responsible for some of the earliest published work on SBS, and recognize the fundamental challenge that any method based on this approach must address before tangible progress to a practical system can be made. That is, to identify combinations of appropriately modified nucleoside triphosphates that will be accepted, efficiently and with high fidelity, by suitably mutated DNA replicating enzymes. Consequently, this proposal features a strong synthetic chemistry component featuring two laboratories focused on the preparing nucleoside triphosphates with fluorescent, labile 3'-protecting groups. It also describes molecular biology to produce relatively large libraries of mutated polymerases. Even though the numbers of modified enzymes generated is high, the mutations will focus on key structural regions to maximize the chances of finding suitable systems. This molecular biology component is coupled with a combinatorial screen to rapidly identify suitable enzyme/modified dNTP pairs. Once suitable combinations are identified, then the SBS methodology will be implemented using high-density arrays that, uniquely, orientate oligonucleotides in the desired 5'-> 3' direction. This core technology fits into a broader, comprehensive research plan encompassing microfluidics for sample manipulation and delivery of the DNA to the SBS system, fluorescent imaging via our proprietary Pulse-Multiline Excitation (PME) system, computational methods for identifying an optimal tiling path and thermodynamic properties of oligonucleotides across whole chromosomes, and informatics to process and store the data generated. The overall goal is, by the end of year three, to complete sequencing of chromosomes 3, 12 & X, which cover approximately 0.5 gigabases and would lay the foundation for whole genome sequencing.