Direct Single Base-Pair Real-Time DNA Methylation Sequencing

描述（由申请人提供）：在该计划中，我们将在实时DNA测序过程中实现直接DNA甲基化分析，而无需亚硫酸氢盐转化。我们将通过定制Pacific Biosciences的高通量单分子实时（SMRT）DNA测序技术来实现这一目标，该技术用于检测由于DNA模板中存在5-甲基胞嘧啶（5 mC）而改变的DNA聚合酶动力学。此外，我们将开发五碱基SMRT测序，其中第5个碱基表示原始样品中存在5 mC。我们提出的技术提供了几个优势，目前的甲基化分析技术。例如，无需亚硫酸氢盐转化和DNA扩增的直接甲基化测序将减少样品制备所需的时间和精力。此外，通过SMRT测序（几种内切酶）提供的长读段将使得能够在高度重复的基因组区域中进行甲基化分析。Pacific Biosciences在开发这项技术方面具有独特的优势，因为我们现有的SMRT测序仪器和聚合酶工程资源基础设施。该计划将在下一个财政季度内创造几个新的就业机会，如果成功的话，随着太平洋生物科学公司向更广泛的研究界提供技术，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展和疾病过程（如癌症）生物学中一个越来越重要的方面。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率甲基化分析的技术，而无需亚硫酸氢盐转化。直接确定基因组DNA甲基化模式的能力将大大加快并降低旨在阐明其在人类健康中作用的项目的成本。

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.