Optimization of Nanopore Genomic DNA Sequencing

描述（由申请人提供）：本提案涉及用于基因组DNA片段单分子测序的酶、孔和计算方法的优化。它基于我们在UCSC的小组实施的经过验证的纳米孔设备。该装置由一个传感器组成，该传感器在进行性酶马达推进DNA链时接触并检查捕获的DNA链内的每个核苷酸。虽然纳米孔测序的总体目标是在非常长的链上从头读取，但在这里，我们还将关注基础研究中重要的生物体（小鼠，E. coli和Arabidopsis）和医疗保健（人类）。我们专注于从头测序和重测序有两个原因：1）生物DNA的纳米孔测序尚未公开记录。因此，参考标准的纳米孔重测序是社区接受所需的，并且重要的是，揭示影响从头测序准确性的技术中的弱点; 2）本申请中的纳米孔重测序意味着直接阅读基因组DNA，因此将包括表观遗传修饰。这将是对研究界的一个直接的、重要的贡献。

DESCRIPTION (provided by applicant): This proposal concerns optimization of enzymes, pores, and computational methods for single molecule sequencing of genomic DNA fragments. It is based on a proven nanopore device implemented by our group at UCSC. This device is comprised of a sensor that touches and examines each nucleotide within a captured DNA strand as a processive enzyme motor advances the strand. Although the overall goal of nanopore sequencing is de novo reads on very long strands, here we will also focus on resequencing of DNA from organisms important in basic research (mouse, E. coli & Arabidopsis) and in healthcare (human). We are focusing on both de novo and resequencing for two reasons: 1) nanopore sequencing of biological DNA has not been documented publicly. Therefore, nanopore resequencing of reference standards is required for community acceptance, and, importantly, to reveal weaknesses in the technology that impact de novo sequencing accuracy; 2) nanopore resequencing in this application means reading genomic DNA directly and therefore will include epigenetic modifications. This would be an immediate, important contribution to the research community.