Highly sensitive amplicon-based RNA quantification by Ion Torrent Proton semiconductor sequencing

Ulrich Certa Hoffmann-La Roche AG, Switzerland

Abstract
The phenotype of a living cell is determined by its pattern of active signaling networks. Thus, a subset of the transcriptome can be used to define the “molecular phenotype” based on gene expression analysis. Digital amplicon based RNA quantification by deep sequencing allows multiparallel gene expression analysis for molecular phenotypic screens as a novel tool to monitor the state of biological systems. We show that the activity of signaling networks can be assessed based on a set of established key regulators and expression targets rather than the entire transcriptome. We compiled a panel of 917 human pathway reporter genes, representing 154 human signaling and metabolic networks for integrated knowledge- and data-driven understanding of biological processes. We show several examples in which reporter genes deliver an accurate pathway-centric view of the biological system under study. Finally, we show for the first time application of AmpliSeq-RNA for analysis of gene expression of a single cell.