Ultrafast SBS Method for Large-Scale Human Resequencing

识别和了解单核苷酸多态(SNPs)的作用将导致准确的诊断 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。是这样的 技术有望导致预防性治疗，以延缓疾病的发生或进展，而处方 最安全最有效的药物。然而，目前的DNA测序技术速度太慢，成本也太高 来完成这些任务。 在这里，我们建议开发一种具有合成测序(SBS)功能的超高速DNA测序系统。 密度低的寡核苷酸阵列，每个都有大约一百万个引物特征。参与其中的协作团队 在这个项目中负责了一些最早发表的关于SBS的工作，并认识到了 任何基于这种方法的方法都必须解决的挑战，才能在实际系统中取得切实的进展 制造。也就是说，为了识别将被接受的适当修饰的核苷三磷酸盐的组合， 通过适当突变的DNA复制酶，高效且高保真。因此，这项提议的特点是 强合成化学成分，具有两个实验室，专注于制备核苷三磷酸盐 带有荧光的、不稳定的3‘-保护基团。它还描述了分子生物学产生相对较大的 突变聚合酶。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是 与组合筛选相结合以快速鉴定合适的酶/修饰dNTP对。一旦合适 确定组合后，将使用高密度阵列实施SBS方法， 将寡核苷酸定向到所需的5‘-3’方向。这项核心技术适合更广泛、全面的 研究计划包括用于样品处理和将DNA输送到SBS系统的微流体， 通过我们专有的脉冲-多线激发(PME)系统的荧光成像，计算方法 确定跨整个染色体的最佳平铺路径和寡核苷酸的热力学性质，以及 信息学来处理和存储生成的数据。总体目标是，到第三年年底，完成 染色体3、12和X的测序，覆盖了大约0.5千兆碱基，将为 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.