Genome Sequencing by Natural DNA Synthesis on Amplified DNA Clones

描述(由申请人提供)：我们建议将SBS最成熟的方面与DNA扩增和高速荧光成像的简化方法相结合，以开发和实现一个快速且廉价的基因组重测序和从头测序平台。我们的平台名为“自然合成测序”(NSB)。扩增的DNA分子克隆将通过使用DNA聚合酶和大多数天然核苷酸的合成，通过循环测序进行大规模平行测序。关键是在用合成法进行DNA测序的过程中，将一小部分可切割的荧光标记核苷酸与天然核苷酸一起用于序列检测。荧光标记的核苷酸掺入不仅稀疏，而且荧光部分也将在每个成像步骤后被切割。这将最大限度地减少对延伸DNA模板的自然结构的修改，并确保DNA合成不会受到显著影响。使用这一策略，均聚物区可以被测序，并且可以实现非常长的读取长度。我们提出了一个新的突破性技术的概念，称为自然DNA合成测序(NSB)。我们还展示了其他几个突破性的创新：1)用微阵列原位大规模平行扩增单个DNA分子，并快速组装DNA模板。2)使用自动机来验证和优化新的NSB化学循环测序，通过使用DNA聚合酶和我们将设计和合成的商业可获得的核苷酸和核苷酸进行合成，以便有效地掺入；3)反应与检测的分离，使系统可扩展到用于全基因组测序的非常高密度的阵列。由于可以使用密度高得多的阵列，并且只使用一种酶(DNA聚合酶)，因此需要的试剂要少得多。这将导致产量的显著提高和试剂成本的降低。4)实现了双桶配对策略和从头序列拼接新算法。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重新测序，用于生物医学研究和个性化医学。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(NSB)。我们将把简化的基因组规模DNA扩增方法与新的测序化学相结合，设计一个超快、低成本的人类基因组测序平台，以便为生物医学和个性化医学应用进行单个人类基因组的常规测序。

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.