Direct Single Base-Pair Real-Time DNA Methylation Sequencing

描述(由申请人提供)：在这个项目中，我们将在实时DNA测序过程中启用直接DNA甲基化分析，而不需要亚硫酸氢盐的转化。我们将通过调整太平洋生物科学公司的高通量单分子实时(SMRT)DNA测序技术来实现这一点，以检测由于DNA模板中存在5-甲基胞嘧啶(5mC)而引起的DNA聚合酶动力学变化。此外，我们还将建立5个碱基的SMRT测序，其中5个碱基表示原始样品中存在5mC。我们建议的技术比目前的甲基化分析技术有几个优点。例如，不需要亚硫酸氢盐转化和DNA扩增的直接甲基化测序将减少样品准备所需的时间和工作量。此外，SMRT测序提供的长读数(几千个碱基)将使高度重复的基因组区域的甲基化分析成为可能。太平洋生物科学公司在开发这项技术方面具有得天独厚的优势，因为我们现有的SMRT测序仪器和聚合酶工程资源的基础设施。该计划将在下一财季创造几个新的就业机会，如果成功，随着太平洋生物科学公司向更广泛的研究社区提供技术，该计划有可能创造数百个新的就业机会。 公共卫生相关性：表观基因组学正在成为发育生物学和癌症等疾病过程中越来越重要的一个方面。我们建议开发一种在实时DNA测序过程中直接进行单碱基对分辨甲基化分析的技术，而不需要亚硫酸氢盐的转化。直接确定基因组DNA甲基化模式的能力将极大地加快和降低旨在阐明其在人类健康中的作用的项目的成本。

DESCRIPTION (provided by applicant): In this program, we will enable direct DNA methylation profiling during real-time DNA sequencing, without bisulfite conversion. We will accomplish this by tailoring Pacific Biosciences' high throughput Single-Molecule Real-Time (SMRT) DNA sequencing technology for the detection of altered DNA polymerase kinetics due to the presence of 5-methylcytosine (5mC) in the DNA template. In addition, we will develop five-base SMRT sequencing, in which the 5th base indicates the presence of 5mC in the original sample. Our proposed technologies offer several advantages over current methylation profiling techniques. For example, direct methylation sequencing without bisulfite conversion and DNA amplification will reduce the time and effort required for sample preparation. In addition, long reads provided by SMRT sequencing (several kilobases) will enable methylation analysis in highly-repetitive genomic regions. Pacific Biosciences is uniquely positioned to develop this technology because of our existing infrastructure of SMRT sequencing instrumentation and polymerase engineering resources. This program will create several new jobs within the next fiscal quarter, and, if successful, has the potential to create hundreds of new jobs as Pacific Biosciences delivers the technology to the broader research community. PUBLIC HEALTH RELEVANCE: Epigenomics is emerging as an ever more important aspect in the biology of development and disease processes such as cancer. We are proposing to develop a technique for direct, single base-pair resolution methylation profiling during real-time DNA sequencing, without bisulfite conversion. The ability to directly determine genomic DNA methylation patterns will dramatically speed up and reduce the cost of projects aiming to illuminate its role in human health.