Development of ONT-cappable-seq to unravel the transcriptional landscape of Pseudomonas phages
RNA sequencing has become the method of choice to study the transcriptional landscape of phage-infected bacteria. However, short-read RNA sequencing approaches generally fail to capture the primary 5′ and 3′ boundaries of transcripts, confounding the discovery of key transcription initiation and ter...
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| Format: | Article |
| Language: | English |
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Elsevier
2022-01-01
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| Series: | Computational and Structural Biotechnology Journal |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S2001037022001908 |
| _version_ | 1797978235441512448 |
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| author | Leena Putzeys Maarten Boon Eveline-Marie Lammens Konstantin Kuznedelov Konstantin Severinov Rob Lavigne |
| author_facet | Leena Putzeys Maarten Boon Eveline-Marie Lammens Konstantin Kuznedelov Konstantin Severinov Rob Lavigne |
| author_sort | Leena Putzeys |
| collection | DOAJ |
| description | RNA sequencing has become the method of choice to study the transcriptional landscape of phage-infected bacteria. However, short-read RNA sequencing approaches generally fail to capture the primary 5′ and 3′ boundaries of transcripts, confounding the discovery of key transcription initiation and termination events as well as operon architectures. Yet, the elucidation of these elements is crucial for the understanding of the strategy of transcription regulation during the infection process, which is currently lacking beyond a handful of model phages. We developed ONT-cappable-seq, a specialized long-read RNA sequencing technique that allows end-to-end sequencing of primary prokaryotic transcripts using the Nanopore sequencing platform. We applied ONT-cappable-seq to study transcription of Pseudomonas aeruginosa phage LUZ7, obtaining a comprehensive genome-wide map of viral transcription start sites, terminators, and complex operon structures that fine-regulate gene expression. Our work provides new insights in the RNA biology of a non-model phage, unveiling distinct promoter architectures, putative small non-coding viral RNAs, and the prominent regulatory role of terminators during infection. The robust workflow presented here offers a framework to obtain a global, yet fine-grained view of phage transcription and paves the way for standardized, in-depth transcription studies for microbial viruses or bacteria in general. |
| first_indexed | 2024-04-11T05:19:45Z |
| format | Article |
| id | doaj.art-a67edec9467e43d1a50baf29da314c32 |
| institution | Directory Open Access Journal |
| issn | 2001-0370 |
| language | English |
| last_indexed | 2024-04-11T05:19:45Z |
| publishDate | 2022-01-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Computational and Structural Biotechnology Journal |
| spelling | doaj.art-a67edec9467e43d1a50baf29da314c322022-12-24T04:52:36ZengElsevierComputational and Structural Biotechnology Journal2001-03702022-01-012026242638Development of ONT-cappable-seq to unravel the transcriptional landscape of Pseudomonas phagesLeena Putzeys0Maarten Boon1Eveline-Marie Lammens2Konstantin Kuznedelov3Konstantin Severinov4Rob Lavigne5Department of Biosystems, Laboratory of Gene Technology, KU Leuven, Leuven 3001, BelgiumDepartment of Biosystems, Laboratory of Gene Technology, KU Leuven, Leuven 3001, BelgiumDepartment of Biosystems, Laboratory of Gene Technology, KU Leuven, Leuven 3001, BelgiumWaksman Institute, Rutgers, The State University, Piscataway, NJ 08854, USAWaksman Institute, Rutgers, The State University, Piscataway, NJ 08854, USADepartment of Biosystems, Laboratory of Gene Technology, KU Leuven, Leuven 3001, Belgium; Corresponding author.RNA sequencing has become the method of choice to study the transcriptional landscape of phage-infected bacteria. However, short-read RNA sequencing approaches generally fail to capture the primary 5′ and 3′ boundaries of transcripts, confounding the discovery of key transcription initiation and termination events as well as operon architectures. Yet, the elucidation of these elements is crucial for the understanding of the strategy of transcription regulation during the infection process, which is currently lacking beyond a handful of model phages. We developed ONT-cappable-seq, a specialized long-read RNA sequencing technique that allows end-to-end sequencing of primary prokaryotic transcripts using the Nanopore sequencing platform. We applied ONT-cappable-seq to study transcription of Pseudomonas aeruginosa phage LUZ7, obtaining a comprehensive genome-wide map of viral transcription start sites, terminators, and complex operon structures that fine-regulate gene expression. Our work provides new insights in the RNA biology of a non-model phage, unveiling distinct promoter architectures, putative small non-coding viral RNAs, and the prominent regulatory role of terminators during infection. The robust workflow presented here offers a framework to obtain a global, yet fine-grained view of phage transcription and paves the way for standardized, in-depth transcription studies for microbial viruses or bacteria in general.http://www.sciencedirect.com/science/article/pii/S2001037022001908Long-read transcriptomicsNanopore sequencingPseudomonas aeruginosaBacteriophagesTranscription regulation |
| spellingShingle | Leena Putzeys Maarten Boon Eveline-Marie Lammens Konstantin Kuznedelov Konstantin Severinov Rob Lavigne Development of ONT-cappable-seq to unravel the transcriptional landscape of Pseudomonas phages Computational and Structural Biotechnology Journal Long-read transcriptomics Nanopore sequencing Pseudomonas aeruginosa Bacteriophages Transcription regulation |
| title | Development of ONT-cappable-seq to unravel the transcriptional landscape of Pseudomonas phages |
| title_full | Development of ONT-cappable-seq to unravel the transcriptional landscape of Pseudomonas phages |
| title_fullStr | Development of ONT-cappable-seq to unravel the transcriptional landscape of Pseudomonas phages |
| title_full_unstemmed | Development of ONT-cappable-seq to unravel the transcriptional landscape of Pseudomonas phages |
| title_short | Development of ONT-cappable-seq to unravel the transcriptional landscape of Pseudomonas phages |
| title_sort | development of ont cappable seq to unravel the transcriptional landscape of pseudomonas phages |
| topic | Long-read transcriptomics Nanopore sequencing Pseudomonas aeruginosa Bacteriophages Transcription regulation |
| url | http://www.sciencedirect.com/science/article/pii/S2001037022001908 |
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