Nanopore direct RNA sequencing applied for characterizing bacterial transcriptomes and epitranscriptomes
Nanopore direct RNA sequencing (DRS) represents a promising approach for comprehensively characterizing bacterial gene expression. However, it has historically been deficient in the yield of mRNA-mapping reads, prohibiting its application for transcriptome-wide RNA modification mapping. To address this limitation, researchers from the City University of Hong Kong have developed a method for pre-processing of bacterial total RNA, based on size selection followed by ribosomal RNA depletion and polyadenylation. Their data, published in Nucleic Acids Research, show this approach to significantly improve sequencing and mapping quality, demonstrating the potential of Nanopore DRS to quickly and systematically characterize bacterial transcriptomes and epitranscriptomes.
Pre-processing of total RNA improves read length, quality, and throughput
To circumvent known challenges for rRNA depletion and enzymatic polyadenylation when conducting DRS with bacterial samples, Tan et al. applied three different pre-processing procedures to total RNAs isolated from E. coli: direct polyadenylation of total RNAs (tot_RNA); rRNA depletion followed by polyadenylation (rd_RNA); and size selection prior to rRNA depletion and polyadenylation (ss&rd_RNA). Following DRS, it was observed that ss&rd_RNA significantly outperformed tot_RNA and rd_RNA in terms of read length, quality, and throughput. The median length of mRNA-mapping reads in E. coli ss&rd_RNA was almost twice that in rd_RNA and had a higher associated Q score. In addition, ss&rd_RNA produced about 1099 Mb data, while tot_RNA and rd_RNA produced around 553 Mb and 58 Mb data, respectively.
Nanopore DRS captures transcript heterogeneity
To assess the ability of Nanopore DRS of ss&rd_RNA to reveal complicated bacterial transcriptome architectures, Tan et al. analyzed datasets generated from differentially processed E. coli RNA samples. ss&rd_RNA again outperformed tot_RNA and rd_RNA, detecting more transcript types and increasing the proportion of genes existing in at least two transcriptional contexts by >20% when compared with the complete list of transcriptional units deposited in RegulonDB. Based on one of the ss&rd_RNA datasets, the boundaries of 225 operons recorded in RegulonDB were extended. Applying the same sequencing strategy to S. aureus RNA extended the boundaries of 89 defined operons. This data supports the use of Nanopore DRS of ss&rd_RNA for detecting intricate transcriptomic features and reliably quantifying protein-coding gene expression.
RNA modifications can be detected from Nanopore DRS data
To enable the application of comparative computational tools for identifying potential modification sites in E. coli and S. aureus genomes, transcriptome-wide modification-free (IVT_neg) RNAs were synthesized via in vitro transcription (IVT). A positive control sample containing m6A in the E. coli IVT transcriptome (IVT_pos) was generated using TriLink’s N6-Methyladenosine-5'-Triphosphate. Analysis of native ss&rd_RNA and IVT_neg samples found considerable overlaps in E. coli, with 21.36% of total predicted sites supported by at least two computational methods. Fewer modification sites could be confidently identified in S. aureus, where the ratio was 6.8%. Tan et al. subsequently identified 75 high-confidence m6A candidates in the E. coli protein-coding transcripts with the aid of MeRIP-Seq.
Conclusion
RNA pre-processing based on size selection followed by rRNA depletion and polyadenylation increased sequencing and mapping qualities for Nanopore DRS. Comparison with a modification-free sample allowed for modification sites to be predicted by computational tools, while the use of MeRIP-Seq aided the identification of high-confidence m6A positions. Together, these data highlight the potential of Nanopore DRS for investigating bacterial transcriptomes and epitranscriptomes.
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Article reference: Tan L, Guo Z, Shao Y, et al. Analysis of bacterial transcriptome and epitranscriptome using nanopore direct RNA sequencing. Nucleic Acids Res. 2024;52(15):8746-8762. https://academic.oup.com/nar/article/52/15/8746/7714457?login=false