Institute of Biochemistry, Genetics and Microbiology, Institute of Microbiology and Archaea Centre, Single-Molecule Biochemistry Lab and Biochemistry Centre Regensburg, University of Regensburg, 93053 Regensburg, Germany.
Regensburg Center of Biochemistry (RCB), University of Regensburg, 93053 Regensburg, Germany.
RNA. 2022 Mar;28(3):400-417. doi: 10.1261/rna.078937.121. Epub 2021 Dec 14.
High-throughput sequencing dramatically changed our view of transcriptome architectures and allowed for ground-breaking discoveries in RNA biology. Recently, sequencing of full-length transcripts based on the single-molecule sequencing platform from Oxford Nanopore Technologies (ONT) was introduced and is widely used to sequence eukaryotic and viral RNAs. However, experimental approaches implementing this technique for prokaryotic transcriptomes remain scarce. Here, we present an experimental and bioinformatic workflow for ONT RNA-seq in the bacterial model organism , which can be applied to any microorganism. Our study highlights critical steps of library preparation and computational analysis and compares the results to gold standards in the field. Furthermore, we comprehensively evaluate the applicability and advantages of different ONT-based RNA sequencing protocols, including direct RNA, direct cDNA, and PCR-cDNA. We find that (PCR)-cDNA-seq offers improved yield and accuracy compared to direct RNA sequencing. Notably, (PCR)-cDNA-seq is suitable for quantitative measurements and can be readily used for simultaneous and accurate detection of transcript 5' and 3' boundaries, analysis of transcriptional units, and transcriptional heterogeneity. In summary, based on our comprehensive study, we show nanopore RNA-seq to be a ready-to-use tool allowing rapid, cost-effective, and accurate annotation of multiple transcriptomic features. Thereby nanopore RNA-seq holds the potential to become a valuable alternative method for RNA analysis in prokaryotes.
高通量测序极大地改变了我们对转录组结构的认识，并在 RNA 生物学领域取得了突破性的发现。最近，基于 Oxford Nanopore Technologies（ONT）的单分子测序平台的全长转录本测序技术被引入并广泛用于真核生物和病毒 RNA 的测序。然而，用于原核转录组的实验方法仍然很少。在这里，我们为细菌模型生物 展示了一种基于 ONT 的 RNA-seq 的实验和生物信息学工作流程，该流程可应用于任何微生物。我们的研究强调了文库制备和计算分析的关键步骤，并将结果与该领域的黄金标准进行了比较。此外，我们全面评估了不同基于 ONT 的 RNA 测序方案的适用性和优势，包括直接 RNA、直接 cDNA 和 PCR-cDNA。我们发现与直接 RNA 测序相比，(PCR)-cDNA 测序具有更高的产量和准确性。值得注意的是，(PCR)-cDNA 测序适用于定量测量，并且可以很容易地用于同时准确检测转录本的 5'和 3'边界、转录单元分析和转录异质性。总之，基于我们的综合研究，我们表明纳米孔 RNA-seq 是一种即用型工具，可快速、经济高效且准确地注释多个转录组特征。因此，纳米孔 RNA-seq 有可能成为原核生物 RNA 分析的一种有价值的替代方法。
