Direct RNA Sequencing Unfolds the Complex Transcriptome of Vibrio parahaemolyticus.

Conventional bacterial genome annotation provides information about coding sequences but ignores untranslated regions and operons. However, untranslated regions contain important regulatory elements as well as targets for many regulatory factors, such as small RNAs. Operon maps are also essential for functional gene analysis. In the last decade, considerable progress has been made in the study of bacterial transcriptomes through transcriptome sequencing (RNA-seq). Given the compact nature of bacterial genomes, many challenges still cannot be resolved through short reads generated using classical RNA-seq because of fragmentation and loss of the full-length information. Direct RNA sequencing is a technology that sequences the native RNA directly without information loss or bias. Here, we employed direct RNA sequencing to annotate the Vibrio parahaemolyticus transcriptome with its full features, including transcription start sites (TSSs), transcription termination sites, and operon maps. A total of 4,103 TSSs were identified. In comparison to short-read sequencing, full-length information provided a deeper view of TSS classification, showing that most internal and antisense TSSs were actually a result of gene overlap. Sequencing the transcriptome of V. parahaemolyticus grown with bile allowed us to study the landscape of pathogenicity island Vp-PAI. Some genes in this region were reannotated, providing more accurate annotation to increase precision in their characterization. Quantitative detection of operons in V. parahaemolyticus showed high complexity in some operons, shedding light on a greater extent of regulation within the same operon. Our study using direct RNA sequencing provides a quantitative and high-resolution landscape of the V. parahaemolyticus transcriptome.