Development of Electron Microscopy-based Nucleic Acid Polymer Sequencing

描述（由申请人提供）：我们的目标是为开发基于重原子标记核酸聚合物的单原子分辨率透射电子显微镜（TEM）的超低成本、超快速核酸聚合物测序技术提供全面的基础。我们的特殊方法是基于用重原子选择性标记碱基的高分子量ssDNA分子的超密集（3纳米链到链间距）平行阵列的TEM成像。这将允许读取长度至少~ 150kb，可能高达2- 4mb或更多，没有高度重复DNA带来的特殊困难。通过适当的优化、自动化和规模化，以及超出本提案范围的进一步资助，该技术（“TEM测序”）将有可能使人类基因组测序以显著更低的成本、更快的速度和共识准确性/完整性比其他拟议的第三代测序方法。我们的项目将包括优化我们的新型ssDNA阵列沉积方案，改善成像条件和底物质量，以及随后设计和构建原型TEM测序系统，我们希望通过提供人类参考基因组组装来展示该方法的潜力，我们相信由于该方法固有的极端读取长度和高覆盖率，该方法可能具有前所未有的一致性准确性和完整性。

DESCRIPTION (provided by applicant): We aim to provide a comprehensive foundation for development of an ultra-low-cost, ultra-fast nucleic acid polymer sequencing technology based on single-atom resolution transmission electron microscopy (TEM) of heavy atom-labeled nucleic acid polymers. Our particular approach is based on TEM imaging of ultra-dense (3 nm strand-to-strand spacing) parallel arrays of high molecular weight ssDNA molecules labeled base- selectively with heavy atoms. This will allow read lengths of at least ~150 kb and potentially as much as 2-4 Mb or more, with no special difficulties posed by highly repetitive DNA. With appropriate optimization, automation, and scaling, and with further funding beyond the scope of this proposal, this technology ("TEM sequencing") will potentially enable human genome sequencing at significantly lower cost and with much greater speed and consensus accuracy/completeness than other proposed third- generation sequencing approaches. Our project will involve the optimization of our novel ssDNA array deposition protocol, improvement of imaging conditions and substrate quality, and subsequent design and building of a prototype TEM sequencing system with which we hope to demonstrate the approach's potential by delivering a human reference genome assembly that we believe may possess unprecedented consensus accuracy and completeness due to the inherently extreme read lengths and high coverage enabled by the approach. PUBLIC HEALTH RELEVANCE: The pace and impact of biomedical research on improving human health may be greatly increased by the development of ultra-low-cost, ultra-high-quality genome sequencing. Our electron microscopy-based approach employs preparation and readout unbiased by sequence content with extremely long read lengths (at least 150,000 bases and potentially as much as 2-4 Mb), suggesting that nearly gapless assemblies will be achievable, shedding light on previously unassembled long repetitive regions and structural variations with potentially important roles in complex disease. Furthermore, our models indicate that TEM sequencing may enable sequencing of whole human genomes to >99.9999% consensus accuracy and completeness in <10 minutes/genome, at a cost of <$100, and thus its realization may have a broad, near- term, lasting impact on biomedical research. )