Discovery of Posttranscriptional Regulatory RNAs Using Next Generation Sequencing Technologies

Next generation sequencing (NGS) has revolutionized the way by which we engineer metabolism by radically altering the path to genome-wide inquiries. This is due to the fact that NGS approaches offer several powerful advantages over traditional methods that include the ability to fully sequence hundreds to thousands of genes in a single experiment and simultaneously detect homozygous and heterozygous deletions, alterations in gene copy number, insertions, translocations, and exome-wide substitutions that include “hot-spot mutations.” This chapter describes the use of these technologies as a sequencing technique for transcriptome analysis and discovery of regulatory RNA elements in the context of three main platforms: Illumina HiSeq, 454 pyrosequencing, and SOLiD sequencing. Specifically, this chapter focuses on the use of Illumina HiSeq, since it is the most widely used platform for RNA discovery and transcriptome analysis. Regulatory RNAs have now been found in all branches of life. In bacteria, noncoding small RNAs (sRNAs) are involved in highly sophisticated regulatory circuits that include quorum sensing, carbon metabolism, stress responses, and virulence (Gorke and Vogel, Gene Dev 22:2914–2925, 2008; Gottesman, Trends Genet 21:399–404, 2005; Romby et al., Curr Opin Microbiol 9:229–236, 2006). Further characterization of the underlying regulation of gene expression remains poorly understood given that it is estimated that over 60% of all predicted genes remain hypothetical and the 5′ and 3′ untranslated regions are unknown for more than 90% of the genes (Siegel et al., Trends Parasitol 27:434–441, 2011). Importantly, manipulation of the posttranscriptional regulation that occurs at the level of RNA stability and export, trans-splicing, polyadenylation, protein translation, and protein stability via untranslated regions (Clayton, EMBO J 21:1881–1888, 2002; Haile and Papadopoulou, Curr Opin Microbiol 10:569–577, 2007) could be highly beneficial to metabolic engineering.