Development of Electron Microscopy-based Nucleic Acid Polymer Sequencing

描述(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透射电子显微镜的超低成本、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地标记碱基的高分子量单链DNA分子的超高密度(3 nm链到链间距)平行阵列的电子显微镜成像。这将允许至少~150kb的读取长度，并可能高达2-4Mb或更多，高度重复的DNA不会造成特殊的困难。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金投入，这项技术(“瞬变电子显微镜测序”)将有可能使人类基因组测序比其他拟议的第三代测序方法更低的成本、更快的速度和一致的准确性/完整性。我们的项目将涉及我们新型的单链DNA阵列沉积方案的优化，成像条件和衬底质量的改善，以及随后设计和建造的原型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，我们认为由于该方法固有的极端读取长度和高覆盖率，我们相信该方法可能具有前所未有的共识准确性和完整性。 公共卫生相关性：超低成本、超高质量基因组测序的发展可能会大大提高生物医学研究改善人类健康的速度和影响。我们基于电子显微镜的方法使用了不受序列内容偏差的制备和读出，具有极长的读取长度(至少150,000个碱基，可能多达2-4 Mb)，这表明几乎没有间隙的组装将是可以实现的，从而揭示了以前未组装的长重复区域和结构变异，这些区域和结构变异在复杂疾病中具有潜在的重要作用。此外，我们的模型表明，电子显微镜测序可以使整个人类基因组测序在10分钟内达到99.9999的共识准确率和完整性，花费100美元，因此它的实现可能会对生物医学研究产生广泛的、短期的、持久的影响。)

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. )