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

这项提议的目标是开发一种基因组测序技术。在R21阶段， 项目中，我们提出了一种制作亚微米高密度井阵列的方法 订购单个纳米颗粒和DNA分子的尺寸。我们将演示原则证明 提出了一种新的DNA测序方法，称为单分子连接测序(SM-SBL)。多过 通过连接循环测序，10亿个DNA分子可以大规模并行测序 使用可光裂解的可逆寡核苷酸探针。因为模板的两端都可以用 SM-SBL，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 使用这种强大的技术进行从头测序的可行性，既有实验上的，也有 从计算上讲。在项目的R33阶段，我们建议进一步改进SM-SBL方法 通过开发一种用荧光纳米颗粒编码寡核苷酸探针的策略 每个测序周期读取3个碱基。纳米粒子将被编码成多个 通过将不同摩尔比的荧光分子嵌入到 纳米粒子。还提出了一种高通量荧光成像的新方法。四带通行证 滤光片和分束器将与能够超快波长选择的光源相结合，用于 荧光成像。微制造技术将被用来对目标分子进行排序，以建立标记 并将分子的间距与光学成像系统相匹配，从而实现最大数据 吞吐量。将开发一个高度集成的基因组规模测序系统。算法和 还将开发从配对末端测序数据进行基因组序列拼接的软件。这部小说 这项技术将使从单个DNA分子的配对末端进行DNA测序成为可能 并行过程，允许快速和低成本的新基因组测序。一旦开发出来，这种 技术将使个人基因组的常规测序成为可能，从而使生物医学研究发生革命性的变化 和医疗保健。

The objective of this proposal is to develop a technologyfor 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 sequenced with SM-SBL, high genome coverage with paired-end information can be obtained. We proposeto 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 probeswith 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 fluorescenceimaging 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-scalesequencing will be developed. Algorithms and software for genome sequence assembly from paired-end sequencedata 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-costde novo genome sequencing. Once developed, this kind of technology will enable routine sequencing of individual human genomes, revolutionizing biomedical research and healthcare.