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

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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.