Direct Real-Time Transcriptome Sequencing

描述（由申请人提供）：在这个项目中，我们将通过将太平洋生物科学公司的高通量单分子实时（SMRT） DNA测序应用于RNA依赖的聚合酶，使SMRT RNA测序可以在同一仪器上进行，从而实现RNA模板的直接测序。SMRT测序提供长读取（目前为3kb，预计到项目结束时将增加到10kb）。这将对转录组的分析产生重要影响，对医学生物学的所有领域都至关重要。例如，它将避免cDNA转换及其相关的偏差，并提供全面检查替代剪接形式的能力，而不是像目前的方法那样通过输入结构。由于现有的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3个新的就业机会，在商业化的时候，有可能在太平洋生物科学公司提供这项技术，并在更广泛的社区应用它，创造数百个就业机会。1

DESCRIPTION (provided by applicant): In this program, we will enable direct sequencing from RNA templates by adapting Pacific Biosciences' high throughput Single-Molecule Real-Time (SMRT) DNA sequencing to an RNA-dependent polymerase so SMRT RNA sequencing can be performed on the same instrument. SMRT sequencing provides long reads (currently 3kb and expected to increase to 10kb by the end of the program). This will have important implications to analysis of transcriptomes, crucial to all areas of medical biology. For example it will avoid cDNA conversion and its associated biases and provide the ability to examine alternative splice forms in their entirely rather than by imputed structure as with current methods. Pacific Biosciences is uniquely positioned to develop this technology because of the existing infrastructure of SMRT sequencing instrumentation and polymerase engineering resources. This program will create 3 new jobs within the next fiscal quarter, and at the point of commercialization, has the potential to create hundreds of jobs at Pacific Biosciences in delivering this technology and still more in the broader community applying it. 1 PUBLIC HEALTH RELEVANCE: The proposed research will improve researchers' ability to rapidly and cost-efficiently determine all genes that are expressed for protein synthesis in a given cell by sequencing these RNA messages. Current methods require conversion of RNA to DNA and look at fragments much smaller than the genes being studied. This research will lead to a method that allows RNA to be sequenced directly, without the degradation caused by conversion to DNA and allowing direct reading of gene-sized pieces. These two abilities will improve the quality of results; reduce the cost of studies that could be performed today, and enable studies that were previously impossible. The result will be new insights into vital processes such as development and regeneration as well as disease processes such as cancer.