Genome Sequencing by Ligation Using Nano-Arrays of Single DNA Molecules

描述（由申请人提供）：本提案的目的是开发基因组测序技术。在该项目的R21阶段，我们建议开发一种用于制造具有亚微米尺寸的高密度威尔斯阵列的方法，用于订购单个纳米颗粒和DNA分子。我们将展示一种新的DNA测序方法的原理证明，称为连接单分子测序（SM-SBL）。超过十亿个单独的DNA分子可以通过使用可光裂解的可逆寡核苷酸探针连接的循环测序来大规模并行测序。由于模板的两端都可以用SM-SBL测序，因此可以获得具有配对末端信息的高基因组覆盖率。我们建议调查从头基因组测序的可行性，使用这种强大的技术，实验和计算。在该项目的R33阶段，我们建议通过开发一种策略来进一步改进SM-SBL方法，该策略用于编码具有荧光纳米颗粒的64个寡核苷酸探针，用于每个测序循环阅读3个碱基。通过将不同摩尔比的荧光分子嵌入纳米颗粒中，纳米颗粒将被编码为具有多个波长和强度水平的组合。提出了一种新的高通量荧光成像方法。四带通滤波器和分束器将与能够超快波长选择的光源组合用于荧光成像。微加工技术将用于对目标分子进行排序，以建立标志柱，并使分子的间距与光学成像系统相匹配，从而实现最大的数据吞吐量。将开发一个高度集成的基因组规模测序系统。还将开发从双端序列数据进行基因组序列组装的算法和软件。这项新技术将使单个DNA分子的配对末端的DNA测序在一个非常大的并行过程中，允许快速和低成本的从头基因组测序。一旦开发出来，这种技术将使人类个体基因组的常规测序成为可能，从而彻底改变生物医学研究和医疗保健。

DESCRIPTION (provided by applicant): The objective of this proposal is to develop a technology for genome sequencing. In the R21 phase of the project, we propose to develop a method for fabricating high-density arrays of wells with sub-micrometer dimensions for ordering single nanoparticles and DNA molecules. We will demonstrate the proof-of-principle of a new method for DNA sequencing called single molecule sequencing by ligation (SM-SBL). More than one billion individual DNA molecules can be sequenced in massive parallel by cyclic sequencing by ligation using photocleavable reversible oligonucleotide probes. Since both ends of a template can be sequenced with SM-SBL, high genome coverage with paired-end information can be obtained. We propose to investigate the feasibility of de novo genome sequencing using this powerful technology, both experimentally and computationally. In the R33 phase of the project, we propose to improve the SM-SBL method even further by developing a strategy for encoding the 64 oligonucleotide probes with fluorescence nanoparticles for reading 3 bases per sequencing cycle. The nanoparticles will be encoded with a combination of multiple wavelengths and intensity levels by embedding the fluorescent molecules in different molar ratios into the nanoparticles. A new method for high throughput fluorescence imaging is also proposed. A quad-bandpass filter and beamsplitter will be combined with a light source capable of ultra-fast wavelength selection for fluorescence imaging. Microfabrication techniques will be used to order the target molecules, to build in sign posts and to match the spacing of the molecules to the optical imaging system allowing for maximum data throughput. A highly integrated system for genome-scale sequencing will be developed. Algorithms and software for genome sequence assembly from paired-end sequence data will also be developed. The novel technology will enable DNA sequencing from paired-ends of single DNA molecules in an extremely massive parallel process, allowing rapid and low-cost de novo genome sequencing. Once developed, this kind of technology will enable routine sequencing of individual human genomes, revolutionizing biomedical research and healthcare.