Optimization of Nanopore Genomic DNA Sequencing

描述（由申请人提供）：该提案涉及基因组DNA片段单分子测序的酶，孔和计算方法的优化。它是基于我们在UCSC的团队实现的一个成熟的纳米孔设备。该装置由一个传感器组成，当一个进程酶马达推进DNA链时，该传感器触摸并检查捕获的DNA链中的每个核苷酸。虽然纳米孔测序的总体目标是在非常长的链上重新读取，但在这里，我们也将重点关注在基础研究（小鼠，大肠杆菌和拟南芥）和医疗保健（人类）中重要生物体的DNA重测序。我们关注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，需要对参考标准进行纳米孔重测序，以获得社区认可，并且重要的是，揭示影响从头测序准确性的技术弱点；2)纳米孔重测序在这个应用中意味着直接读取基因组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.