Ultrafast SBS Method for Large-Scale Human Resequencing

识别和理解单核苷酸多态性（SNPs）的作用将有助于准确诊断 遗传性疾病状态、风险因素的确定以及患者代谢特征的表征。等 技术有望导致预防性治疗，以延缓疾病的发作或进展， 最安全最有效的药物然而，目前的DNA测序技术过于缓慢和昂贵 对于这些任务。 在这里，我们建议开发一个超快的DNA测序系统，其特点是合成测序（SBS）在高分辨率上。 密度寡核苷酸阵列，每个具有大约一百万个引物特征。参与的合作团队 在这个项目中，我负责SBS最早发表的一些工作，并认识到基本的 任何基于这种方法的方法在实际系统取得切实进展之前都必须解决的挑战， 进行了也就是说，为了鉴定将被接受的适当修饰的核苷三磷酸的组合， 通过适当突变的DNA复制酶高效且高保真地进行。因此，本提案的特点是： 强大的合成化学成分，包括两个专注于制备核苷三磷酸的实验室 具有荧光的不稳定的3 '-保护基团。它还描述了分子生物学，以产生相对较大的文库， 突变的聚合酶。尽管产生的修饰酶的数量很高，但突变将集中在 关键结构区域，以最大限度地找到合适的系统的机会。这个分子生物学组件是 结合组合筛选以快速鉴定合适的酶/修饰的dNTP对。一旦合适 组合被识别，然后SBS方法将使用高密度阵列来实施，独特地， 使寡核苷酸以所需的5 ′-3 ′方向取向。这一核心技术符合更广泛、全面的 研究计划包括用于样品操作和将DNA递送到SBS系统的微流体， 通过我们专有的脉冲多线激发（PME）系统进行荧光成像， 识别跨整个染色体的寡核苷酸的最佳平铺路径和热力学性质，以及 信息处理和存储生成的数据。总体目标是，到第三年年底， 染色体3，12和X的测序，其中涵盖约0.5千兆碱基，并将奠定基础， 全基因组测序

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.