Genome Sequencing by Natural DNA Synthesis on Amplified DNA Clones

通过对扩增的 DNA 克隆进行天然 DNA 合成进行基因组测序

• 批准号: 7676229
• 负责人: XIAOHUA HUANG
• 金额: $ 61.76万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-08-19 至 2012-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7676229
• 关键词: Affect; Algorithms; Applied Research; Base Pairing; Biological Sciences; Biomedical Research; Cells; Chemistry; Cleaved cell; Computer software; Cyclic Nucleotides; DNA; DNA Resequencing; DNA Sequence; DNA amplification; DNA biosynthesis; DNA-Directed DNA Polymerase; Detection; Disulfides; Engineering; Ensure; Enzymes; Fluorescent Dyes; Genome; Glass; Goals; Human Genome; Image; In Situ; Individual; Label; Length; Medicine; Methods; Microspheres; Modification; Molecular Cloning; Nucleotides; Polymers; Process; Reaction; Reading; Reagent; Research; Running; Speed; Structure; Surface; Synthesis Chemistry; System; Technology; base; cost; density; design; design and construction; fluorescence imaging; genome sequencing; innovation; monolayer; programs

DESCRIPTION (provided by applicant): We propose to combine the best proven aspects of SBS with streamlined methods for DNA amplification and high-speed fluorescence imaging to develop and implement a platform for rapid and inexpensive genome resequencing and de novo sequencing. Our platform is called "Natural Sequencing by Synthesis" (nSBS). Amplified DNA molecular clones will be sequenced in massive parallel by cyclic sequencing by synthesis using DNA polymerases and mostly natural nucleotides. The key is to use a small percentage of a cleavable fluorescently-labeled nucleotide along with the natural nucleotide in the cyclic base-by-base DNA sequencing by synthesis process for sequence detection. Not only will the fluorescently-labeled nucleotide incorporation be sparse but the fluorescent moiety will also be cleaved off after each imaging step. This will minimize the modification of the natural structure of the extending DNA template and ensure that DNA synthesis will not be significantly affected. With this strategy, homopolymer tracts can be sequenced and very long read lengths can be achieved. We present a concept for a new breakthrough technology called natural DNA sequencing by synthesis (nSBS). We also present several other breakthrough innovations: 1) In situ massive parallel amplification of single DNA molecules with micro fabricated arrays and rapid assembly of DNA templates. 2) The usage of an automaton to validate and optimize the new nSBS chemistry for cyclic sequencing by synthesis using DNA polymerases and commercially available nucleotides and nucleotides we will design and synthesize for efficient incorporation; 3) The decoupling of the reaction from detection to make the system scalable to very high-density arrays for whole genome sequencing. Since much higher density arrays can be used and only one enzyme (DNA polymerase) will be used, much less reagent will be needed. This will result in dramatic improvement of throughput and reduction in reagent cost. 4) The implementation of a double barrel paired-end strategy and new algorithms for de novo sequence assembly. In the long run this technology will have a great potential to enable very accurate re-sequencing and de novo sequencing of genomes at high speed and much lower cost for biomedical research and personalized medicine. PROJECT HEALTH RELEVANCE We propose to develop a breakthrough DNA sequencing technology called DNA sequencing by natural DNA synthesis (nSBS). We will combine streamlined methods for genome-scale DNA amplification with the new sequencing chemistry to engineer a sequencing platform for ultra-fast and low-cost human genome sequencing so that routine sequencing of individual human genomes can be performed for biomedical applications and personalized medicine.

描述（由申请人提供）：我们建议将SB的最佳验证方面与简化的DNA扩增方法和高速荧光成像相结合，以开发和实施一个平台，以快速且廉价的基因组重新配置和从头测序。我们的平台称为“合成的自然测序”（NSB）。通过使用DNA聚合酶和大部分天然核苷酸通过合成，通过合成，将在大规模平行中对扩增的DNA分子克隆进行测序。关键是要在循环基碱基DNA测序中通过合成过程中使用一小部分可切除的荧光标记的核苷酸以及天然核苷酸来进行序列检测。荧光标记的核苷酸掺入不仅会稀疏，而且在每个成像步骤之后，荧光部分也会裂解。这将最大程度地减少扩展DNA模板的自然结构的修饰，并确保不会显着影响DNA合成。通过这种策略，可以测序均聚物区，并可以实现很长的读取长度。我们提出了一种新的突破性技术的概念，称为合成（NSB）的天然DNA测序。我们还提出了其他几项突破性创新：1）与微型制造阵列和DNA模板快速组装的单个DNA分子的原位平行扩增。 2）使用自动机验证和优化新的NSBS化学，使用DNA聚合酶以及市售的核苷酸和核苷酸，以通过合成，我们将设计和合成以有效掺入； 3）从检测中反应的解耦，以使系统可扩展到非常高密度的阵列，以进行整个基因组测序。由于可以使用更高的密度阵列，并且仅使用一种酶（DNA聚合酶），因此需要的试剂要少。这将导致试剂成本的吞吐量和降低的急剧改善。 4）实施双桶配对策略和新序列组件的新算法。从长远来看，这项技术将具有巨大的潜力，可以使基因组以高速进行非常准确的重新测序和从头开始，并且生物医学研究和个性化医学的成本要低得多。 我们建议通过自然DNA合成（NSB）开发一种称为DNA测序的突破性DNA测序技术。我们将结合用于基因组尺度DNA扩增的方法与新的测序化学方法，以设计一个测序平台，用于超快速和低成本的人类基因组测序，以便可以对单个人类基因组进行常规测序，以用于生物医学应用和个性化药物。