Analyzing Multiplexed Amplicons for Advanced Sequencing

尽管人类基因组的测序工作已于2002年基本完成，但仍有生产DMA序列数据的巨大需求。可进行测序的生物体和个体的数量仍然受到当今桑格测序方法耗时长、复杂性高和成本过高的显著限制。目前正在进行大量工作，以创造新的化学方法，在不使用电泳法的情况下读出DNA链的碱基；然而，为这些测序系统提供数据的样品制备过程尚未完全确定和优化。许多先进的测序方法需要数百万组扩增的DNA片段，每组都由数百万个相同的片段组成。这些组件连接到坚固的支撑件上，然后 被处理以读出序列。我们建议使用新型的超高密度微机械平板作为容器来制备用于测序的纳升尺度的反应。这种制备通常包括DNA片断、扩增、浓缩和固相载体连接步骤，这些步骤导致芯片中有大量样本准备进行测序。目前提出的使用Polonies或珠子的样品制备方法可能需要大量步骤才能成功，研究人员可能没有自由实践某些受保护的基于珠子的技术。拟议的第一阶段项目将包括 演示几个片段物种的DNA扩增，并确定从先进的合成测序系统的单个点可以合理分析多少不同的片段。在每个地点分析多种物种的能力可以提高产量，并显著简化样品准备步骤。第一阶段里程碑的成功完成以及随后的第二阶段和第三阶段项目的完成将导致开发一种超高通量原型系统，该系统可以生产成本效益高的高密度芯片，与先进的DNA测序仪器一起使用。最终，这种下一代的样本准备和测序 技术可以产生几个数量级的DNA序列，比现有系统更便宜、更快，这有助于使个性化治疗的承诺成为现实。

Although the sequence of the human genome was substantially completed in 2002, there is still a huge need for the production of DMA sequence data. The number of organisms and individuals which may be sequenced is still significantly constrained by the lengthy time, high complexity and exorbitant costs of today's Sanger sequencing methods. Significant effort is currently going into the creation of new chemistries to read out the bases of DMA strands without the use of electrophoresis; however, the sample preparation processes to feed these sequencing systems has yet to be fully worked out and optimized. Many of the advanced sequencing methods require many millions of sets of amplified DNA fragments, with each set being comprised of millions of identical fragments. These sets are attached to a solid support which is then processed to read out the sequence. We propose the use of novel ultra-high-density micromachined plates as the vessels for preparing nanoliter-scale reactions for sequencing. This preparation generally includes DNA fragmentation, amplification, enrichment and solid-support-attachment steps which result in a chip with a large number of samples ready to be sequenced. Currently proposed sample preparation methods using polonies or beads may require a large number of steps to succeed and researchers may not have the freedom to practice certain protected bead-based technologies. The proposed Phase I project will include the demonstration of DNA amplification of several fragment species and a determination of how many different fragments may be reasonably analyzed from a single spot of an advanced sequencing by synthesis system. The ability to analyze multiple species in each spot could improve throughput and significantly simplify-sample preparation steps. Successful accomplishment of the Phase I milestones and completion of a subsequent Phase II and Phase III projects will result in the development of an ultra-high throughput prototype system which can produce cost-effective high-density chips for use in conjunction with advanced DNA sequencing instruments. Ultimately, this next generation of sample preparation and sequencing technologies, which can produce DNA sequence several orders of magnitude cheaper and faster than existing systems, can help to make the promise of individualized therapeutics a reality.