Analysis of Transcriptome and Epitranscriptome in Bacteria Using Nanopore Direct RNA Sequencing

In this study, we utilized Nanopore DRS (Direct RNA Sequencing) to analyze the transcriptomes of two representative bacteria: the gram-negative bacterium E. coli and the gram-positive pathogen Staphylococcus aureus (S. aureus). We implemented an explicit pre-processing step for bacterial RNAs prior to Nanopore DRS library preparation, which resulted in a significant improvement in sequencing and mapping quality. By applying this improved approach, we were able to capture more transcriptomic features and obtain more reliable quantitative estimates of gene expression levels. To gain insights into potential RNA modifications in the bacterial transcriptomes, we employed comparative computational methods using modification-free in vitro transcribed (IVT) RNAs as negative controls. Specifically, we utilized MeRIP-Seq (Methylated RNA Immunoprecipitation Sequencing) to assist in the specific prediction of m6A (N6-methyladenosine) modification sites. For DRS ONT sequencing data, it is recommended to download the original fast5 file and base-call again because Nanopore periodically updates its base-calling model and improves accuracy, and the current signal is recorded in the fast5 file.