Genome Sequencing by Natural DNA Synthesis on Amplified DNA Clones

描述（由申请人提供）：我们建议将 SBS 的最佳经过验证的方面与 DNA 扩增和高速荧光成像的简化方法相结合，以开发和实施快速且廉价的基因组重测序和从头测序的平台。我们的平台称为“合成自然测序”(nSBS)。扩增的 DNA 分子克隆将通过使用 DNA 聚合酶和大部分天然核苷酸合成的循环测序进行大规模平行测序。关键是在循环逐碱基 DNA 测序合成过程中使用小比例的可裂解荧光标记核苷酸以及天然核苷酸来进行序列检测。不仅荧光标记的核苷酸掺入会稀疏，而且荧光部分也会在每个成像步骤后被切除。这将最大限度地减少对延伸 DNA 模板自然结构的修饰，并确保 DNA 合成不会受到显着影响。通过这种策略，可以对同聚物束进行测序，并且可以获得非常长的读长。我们提出了一种新突破性技术的概念，称为天然 DNA 边合成边测序 (nSBS)。我们还展示了其他几项突破性创新：1）利用微型阵列和 DNA 模板快速组装对单个 DNA 分子进行原位大规模平行扩增。 2) 使用自动机来验证和优化用于循环测序的新 nSBS 化学，通过使用 DNA 聚合酶和市售核苷酸进行合成，我们将设计和合成核苷酸以实现有效掺入； 3) 将反应与检测分离，使系统可扩展到用于全基因组测序的极高密度阵列。由于可以使用更高密度的阵列并且仅使用一种酶（DNA 聚合酶），因此需要的试剂要少得多。这将显着提高通量并降低试剂成本。 4）双桶双端策略的实现和从头序列组装的新算法。从长远来看，这项技术将具有巨大的潜力，能够以非常准确的速度对基因组进行重测序和从头测序，并以更低的成本进行生物医学研究和个性化医疗。 项目健康相关性 我们建议开发一种突破性的 DNA 测序技术，称为自然 DNA 合成 DNA 测序 (nSBS)。我们将把基因组规模 DNA 扩增的简化方法与新的测序化学相结合，设计一个用于超快速、低成本人类基因组测序的测序平台，以便可以对个体人类基因组进行常规测序，用于生物医学应用和个性化医疗。

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.