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测序期间启用直接DNA甲基化分析，而不需要亚硫酸盐转化。我们将通过定制太平洋生物科学公司的高通量单分子实时（SMRT） DNA测序技术来检测由于DNA模板中存在5-甲基胞嘧啶（5mC）而改变的DNA聚合酶动力学来实现这一目标。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5mC。我们提出的技术与目前的甲基化分析技术相比有几个优势。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制备所需的时间和精力。此外，SMRT测序提供的长读数（数千个碱基）将使高度重复的基因组区域的甲基化分析成为可能。由于我们现有的SMRT测序仪器和聚合酶工程资源的基础设施，太平洋生物科学公司在开发这项技术方面处于独特的地位。该项目将在下一个财政季度创造几个新的工作岗位，如果成功，随着太平洋生物科学公司将这项技术提供给更广泛的研究社区，它有可能创造数百个新的工作岗位。

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.