Improving the Reliability of RNA-seq: Approaching Single-Cell Transcriptomics to Explore Individuality in Bacteria

该项目的主要目标是：i）提高Illumina平台上RNA测序的可靠性;ii）开发一种新的、更灵敏的实验管道，用于对单个细菌细胞进行测序;iii）最后，探索同基因细胞的个体转录组。目前使用的技术需要大量的细菌细胞用于一次测序运行。因此，为了达到单细胞分辨率，需要开发和验证新的文库制备方法和扩增方案。此外，需要应用编码理论方法（条形码）来减少测序过程中不可避免的技术可变性。特别是，我们将开发条形码来改善多重性并减少扩增噪声，否则扩增噪声将隐藏细胞中mRNA数量的生物学可变性。这也需要通过统计分析和适当的实验建立一个全面的RNA-seq通道模型，然后使用新建立的测序程序来探索转录组谱的随机细胞间变异性。我们对随机细胞状态转换现象特别感兴趣，这还没有在全基因组范围内进行研究，并探索转录事件的基本机制，例如，导致转录爆发的机制

The main goals of this project are: i) to improve the reliability of RNA sequencing on Illumina platforms;ii) to develop a new, more sensitive, experimental pipeline for sequencing single bacterial cells;iii) and, finally, to explore the individual transcriptome of isogenic cells. Currently used techniques need a large number of bacterial cells for one sequencing run. Hence, to reach single cell resolution new library preparation approaches and amplification schemes are required, which will be developed and validated. In addition, coding theoretic methods (barcodes) need to be applied to reduce the inevitable technical variability of the sequencing process.In particular, we will develop barcodes to improve multiplexing and to reduce the amplification noise, which otherwise will hide the biological variability in the number of mRNAs in cells. This will also require to establish a comprehensive channel model of RNA-seq using statistical analysis and suitable experiments.The new established sequencing procedure will then be used to explore the stochastic cell-to-cell variability of transcriptomic profiles. We are especially interested in the phenomena of stochastic cell-state switching, which has not yet been studied on a genome wide scale, and to explore basic mechanisms of transcription events, e.g., the mechanisms causing transcriptional bursts.