Genome Sequencing by Natural DNA Synthesis on Amplified DNA Clones

描述（由申请人提供）： 我们建议联合收割机结合SBS的最佳证明方面与简化的DNA扩增和高速荧光成像的方法，以开发和实施一个平台，快速和廉价的基因组重测序和从头测序。我们的平台被称为“合成天然测序”（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。关键是在通过合成进行序列检测的环状逐碱基DNA测序过程中使用小百分比的可裂解荧光标记核苷酸沿着天然核苷酸。不仅荧光标记的核苷酸掺入是稀疏的，而且荧光部分也将在每个成像步骤后被切割掉。这将使延伸DNA模板的天然结构的修饰最小化，并确保DNA合成不会受到显著影响。通过这种策略，可以对均聚物片段进行测序，并且可以实现非常长的读取长度。我们提出了一种新的突破性技术的概念，称为天然DNA合成测序（nSBS）。我们还提出了其他几个突破性的创新：1）原位大规模并行扩增单个DNA分子的微制造阵列和快速组装的DNA模板。2)使用自动机来验证和优化用于通过使用DNA聚合酶和市售核苷酸的合成进行循环测序的新nSBS化学，我们将设计和合成用于有效掺入的核苷酸; 3）将反应与检测解耦，以使系统可扩展到用于全基因组测序的非常高密度的阵列。由于可以使用高得多的密度阵列，并且仅使用一种酶（DNA聚合酶），因此将需要少得多的试剂。这将导致通量的显著提高和试剂成本的降低。4)双桶双端策略的实现和新的从头序列组装算法。从长远来看，这项技术将具有巨大的潜力，能够以高速和低得多的成本对基因组进行非常准确的重新测序和从头测序，用于生物医学研究和个性化医疗。 我们计划开发一种突破性的DNA测序技术，称为自然DNA合成DNA测序（nSBS）。我们将联合收割机结合基因组规模DNA扩增的简化方法和新的测序化学，设计一个用于超快速和低成本人类基因组测序的测序平台，以便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.