Unfolding the Bacterial Transcriptome Landscape Using Oxford Nanopore Technology Direct RNA Sequencing

Current genome annotation ignores important features of the transcriptome, such as untranslated regions and operon maps. RNA sequencing (RNA-seq) helps in identifying such features; however, the fragmentation step of classical RNA-seq makes this task challenging. Long-read sequencing methods, such as that of Oxford Nanopore Technologies (ONT), enable the sequencing of intact RNA molecules. Here, we present a method to annotate the full features of bacterial transcriptomes by combining a modified ONT direct RNA-seq method with our computational pipeline, UNAGI bacteria. The method reveals the full complexity of the bacterial transcriptome landscape, including transcription start sites, transcription termination sites, operon maps, and novel genes.