Department of Infectious Diseases and Public Health, Jockey Club College of Veterinary Medicine and Life Sciences, City University of Hong Kong, Hong Kong, China.
Department of Biomedical Sciences, Jockey Club College of Veterinary Medicine and Life Sciences, City University of Hong Kong, Hong Kong, China.
Nucleic Acids Res. 2024 Aug 27;52(15):8746-8762. doi: 10.1093/nar/gkae601.
Bacterial gene expression is a complex process involving extensive regulatory mechanisms. Along with growing interests in this field, Nanopore Direct RNA Sequencing (DRS) provides a promising platform for rapid and comprehensive characterization of bacterial RNA biology. However, the DRS of bacterial RNA is currently deficient in the yield of mRNA-mapping reads and has yet to be exploited for transcriptome-wide RNA modification mapping. Here, we showed that pre-processing of bacterial total RNA (size selection followed by ribosomal RNA depletion and polyadenylation) guaranteed high throughputs of sequencing data and considerably increased the amount of mRNA reads. This way, complex transcriptome architectures were reconstructed for Escherichia coli and Staphylococcus aureus and extended the boundaries of 225 known E. coli operons and 89 defined S. aureus operons. Utilizing unmodified in vitro-transcribed (IVT) RNA libraries as a negative control, several Nanopore-based computational tools globally detected putative modification sites in the E. coli and S. aureus transcriptomes. Combined with Next-Generation Sequencing-based N6-methyladenosine (m6A) detection methods, 75 high-confidence m6A candidates were identified in the E. coli protein-coding transcripts, while none were detected in S. aureus. Altogether, we demonstrated the potential of Nanopore DRS in systematic and convenient transcriptome and epitranscriptome analysis.
细菌基因表达是一个复杂的过程，涉及广泛的调控机制。随着人们对这一领域兴趣的不断增长，纳米孔直接 RNA 测序（DRS）为快速全面地研究细菌 RNA 生物学提供了一个有前途的平台。然而，目前细菌 RNA 的 DRS 在 mRNA 映射读取的产量上存在不足，尚未被用于全转录组 RNA 修饰图谱绘制。在这里，我们展示了对细菌总 RNA 进行预处理（大小选择，然后去除核糖体 RNA 和多聚腺苷酸化）可以保证测序数据的高通量，并显著增加 mRNA 读取量。通过这种方式，我们为大肠杆菌和金黄色葡萄球菌重建了复杂的转录组结构，并扩展了 225 个已知的大肠杆菌操纵子和 89 个定义明确的金黄色葡萄球菌操纵子的边界。利用未修饰的体外转录（IVT）RNA 文库作为阴性对照，几种基于纳米孔的计算工具全局检测了大肠杆菌和金黄色葡萄球菌转录组中的潜在修饰位点。与基于下一代测序的 N6-甲基腺苷（m6A）检测方法相结合，在大肠杆菌的蛋白编码转录物中鉴定出 75 个高可信度的 m6A 候选者，而在金黄色葡萄球菌中则没有检测到。总之，我们展示了纳米孔 DRS 在系统和便捷的转录组和表转录组分析中的潜力。
