SMRT-Cappable-seq reveals complex operon variants in bacteria

We develop a method SMRT-Cappable-seq that combines the isolation of unfragmented bacterial primary transcripts with the longread sequencing using PacBio. This method allows the identification and phasing of the transcrription start sites and the termination sites, thereby revealing the operon structure and the regulation of gene experession in bacteria. Applied to E.coli, our method results in an unprecedented definition of the transcriptome with 34% of the known operons from RegulonDB database being extended by at least one gene, and identifies a total of 2300 operons from which around 900 are novel. Total RNAs were isolated from E. coli MG1655 cells grown under both M9 minimal and Rich condition. Primary transcripts were capped with desthiobiotin and extracted using streptavidin beads to form Enrich Library. While total RNAs without streptavidin enrichment were used to form Control Library. The RNAs were converted to cDNAs, then the cDNAs were amplified and sequenced using PacBio RSII and Sequel.