Optimization of Nanopore Genomic DNA Sequencing

描述(由申请人提供)：本提案涉及基因组DNA片段的单分子测序的酶、孔和计算方法的优化。它是基于我们在加州大学圣地亚哥分校的团队实施的经过验证的纳米孔设备。这个装置由一个传感器组成，当一个前进的酶马达推进DNA链时，它会触摸和检查捕获的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进行重新读取，但这里我们也将重点放在对基础研究(小鼠、大肠杆菌和拟南芥)和医疗保健(人类)重要的生物的DNA重新测序上。我们专注于从头测序和重新测序，原因有两个：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新测序是社区接受所必需的，重要的是要揭示影响从头测序准确性的技术中的弱点；2)本应用中的纳米孔重新测序意味着直接读取基因组DNA，因此将包括表观遗传修饰。这将是对研究界的直接而重要的贡献。

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.