Adaptable and comprehensive approaches for long-read nanopore sequencing of polyadenylated and non-polyadenylated RNAs
The advent of long-read (LR) sequencing technologies has provided a direct opportunity to determine the structure of transcripts with potential for end-to-end sequencing of full-length RNAs. LR methods that have been described to date include commercial offerings from Oxford Nanopore Technologies (O...
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Frontiers Media S.A.
2024-12-01
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| Series: | Frontiers in Genetics |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fgene.2024.1466338/full |
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| author | Simon Haile Richard D. Corbett Kieran O’Neill Jing Xu Duane E. Smailus Pawan K. Pandoh Anthony Bayega Miruna Bala Eric Chuah Robin J. N. Coope Richard A. Moore Karen L. Mungall Yongjun Zhao Yussanne Ma Marco A. Marra Marco A. Marra Steven J. M. Jones Steven J. M. Jones Andrew J. Mungall |
| author_facet | Simon Haile Richard D. Corbett Kieran O’Neill Jing Xu Duane E. Smailus Pawan K. Pandoh Anthony Bayega Miruna Bala Eric Chuah Robin J. N. Coope Richard A. Moore Karen L. Mungall Yongjun Zhao Yussanne Ma Marco A. Marra Marco A. Marra Steven J. M. Jones Steven J. M. Jones Andrew J. Mungall |
| author_sort | Simon Haile |
| collection | DOAJ |
| description | The advent of long-read (LR) sequencing technologies has provided a direct opportunity to determine the structure of transcripts with potential for end-to-end sequencing of full-length RNAs. LR methods that have been described to date include commercial offerings from Oxford Nanopore Technologies (ONT) and Pacific Biosciences. These kits are based on selection of polyadenylated (polyA+) RNAs and/or oligo-dT priming of reverse transcription. Thus, these approaches do not allow comprehensive interrogation of the transcriptome due to their exclusion of non-polyadenylated (polyA-) RNAs. In addition, polyA + specificity also results in 3′-biased measurements of PolyA+ RNAs especially when the RNA input is partially degraded. To address these limitations of current LR protocols, we modified rRNA depletion protocols that have been used in short-read sequencing: one approach representing a ligation-based method and the other a template-switch cDNA synthesis-based method to append ONT-specific adaptor sequences and by removing any deliberate fragmentation/shearing of RNA/cDNA. Here, we present comparisons with poly+ RNA-specific versions of the two approaches including the ONT PCR-cDNA Barcoding kit. The rRNA depletion protocols displayed higher proportions (30%–50%) of intronic content compared to that of the polyA-specific protocols (5%–8%). In addition, the rRNA depletion protocols enabled ∼20–50% higher detection of expressed genes. Other metrics that were favourable to the rRNA depletion protocols include better coverage of long transcripts, and higher accuracy and reproducibility of expression measurements. Overall, these results indicate that the rRNA depletion-based protocols described here allow the comprehensive characterization of polyadenylated and non-polyadenylated RNAs. While the resulting reads are long enough to help decipher transcript structures, future endeavors are warranted to improve the proportion of individual reads representing end-to-end spanning of transcripts. |
| format | Article |
| id | doaj-art-e269d045c28b4c1d831d718f21bf703b |
| institution | Kabale University |
| issn | 1664-8021 |
| language | English |
| publishDate | 2024-12-01 |
| publisher | Frontiers Media S.A. |
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| series | Frontiers in Genetics |
| spelling | doaj-art-e269d045c28b4c1d831d718f21bf703b2024-12-02T10:53:51ZengFrontiers Media S.A.Frontiers in Genetics1664-80212024-12-011510.3389/fgene.2024.14663381466338Adaptable and comprehensive approaches for long-read nanopore sequencing of polyadenylated and non-polyadenylated RNAsSimon Haile0Richard D. Corbett1Kieran O’Neill2Jing Xu3Duane E. Smailus4Pawan K. Pandoh5Anthony Bayega6Miruna Bala7Eric Chuah8Robin J. N. Coope9Richard A. Moore10Karen L. Mungall11Yongjun Zhao12Yussanne Ma13Marco A. Marra14Marco A. Marra15Steven J. M. Jones16Steven J. M. Jones17Andrew J. Mungall18Canada’s Michael Smith Genome Sciences Centre, BC Cancer, Vancouver, BC, CanadaCanada’s Michael Smith Genome Sciences Centre, BC Cancer, Vancouver, BC, CanadaCanada’s Michael Smith Genome Sciences Centre, BC Cancer, Vancouver, BC, CanadaCanada’s Michael Smith Genome Sciences Centre, BC Cancer, Vancouver, BC, CanadaCanada’s Michael Smith Genome Sciences Centre, BC Cancer, Vancouver, BC, CanadaCanada’s Michael Smith Genome Sciences Centre, BC Cancer, Vancouver, BC, CanadaCanada’s Michael Smith Genome Sciences Centre, BC Cancer, Vancouver, BC, CanadaCanada’s Michael Smith Genome Sciences Centre, BC Cancer, Vancouver, BC, CanadaCanada’s Michael Smith Genome Sciences Centre, BC Cancer, Vancouver, BC, CanadaCanada’s Michael Smith Genome Sciences Centre, BC Cancer, Vancouver, BC, CanadaCanada’s Michael Smith Genome Sciences Centre, BC Cancer, Vancouver, BC, CanadaCanada’s Michael Smith Genome Sciences Centre, BC Cancer, Vancouver, BC, CanadaCanada’s Michael Smith Genome Sciences Centre, BC Cancer, Vancouver, BC, CanadaCanada’s Michael Smith Genome Sciences Centre, BC Cancer, Vancouver, BC, CanadaCanada’s Michael Smith Genome Sciences Centre, BC Cancer, Vancouver, BC, CanadaDepartment of Medical Genetics, University of British Columbia, Vancouver, BC, CanadaCanada’s Michael Smith Genome Sciences Centre, BC Cancer, Vancouver, BC, CanadaDepartment of Medical Genetics, University of British Columbia, Vancouver, BC, CanadaCanada’s Michael Smith Genome Sciences Centre, BC Cancer, Vancouver, BC, CanadaThe advent of long-read (LR) sequencing technologies has provided a direct opportunity to determine the structure of transcripts with potential for end-to-end sequencing of full-length RNAs. LR methods that have been described to date include commercial offerings from Oxford Nanopore Technologies (ONT) and Pacific Biosciences. These kits are based on selection of polyadenylated (polyA+) RNAs and/or oligo-dT priming of reverse transcription. Thus, these approaches do not allow comprehensive interrogation of the transcriptome due to their exclusion of non-polyadenylated (polyA-) RNAs. In addition, polyA + specificity also results in 3′-biased measurements of PolyA+ RNAs especially when the RNA input is partially degraded. To address these limitations of current LR protocols, we modified rRNA depletion protocols that have been used in short-read sequencing: one approach representing a ligation-based method and the other a template-switch cDNA synthesis-based method to append ONT-specific adaptor sequences and by removing any deliberate fragmentation/shearing of RNA/cDNA. Here, we present comparisons with poly+ RNA-specific versions of the two approaches including the ONT PCR-cDNA Barcoding kit. The rRNA depletion protocols displayed higher proportions (30%–50%) of intronic content compared to that of the polyA-specific protocols (5%–8%). In addition, the rRNA depletion protocols enabled ∼20–50% higher detection of expressed genes. Other metrics that were favourable to the rRNA depletion protocols include better coverage of long transcripts, and higher accuracy and reproducibility of expression measurements. Overall, these results indicate that the rRNA depletion-based protocols described here allow the comprehensive characterization of polyadenylated and non-polyadenylated RNAs. While the resulting reads are long enough to help decipher transcript structures, future endeavors are warranted to improve the proportion of individual reads representing end-to-end spanning of transcripts.https://www.frontiersin.org/articles/10.3389/fgene.2024.1466338/fullRNA-seqOxford NanoporecDNAfull-lengthpolyadenylated and non-polyadenylatedlong-read |
| spellingShingle | Simon Haile Richard D. Corbett Kieran O’Neill Jing Xu Duane E. Smailus Pawan K. Pandoh Anthony Bayega Miruna Bala Eric Chuah Robin J. N. Coope Richard A. Moore Karen L. Mungall Yongjun Zhao Yussanne Ma Marco A. Marra Marco A. Marra Steven J. M. Jones Steven J. M. Jones Andrew J. Mungall Adaptable and comprehensive approaches for long-read nanopore sequencing of polyadenylated and non-polyadenylated RNAs Frontiers in Genetics RNA-seq Oxford Nanopore cDNA full-length polyadenylated and non-polyadenylated long-read |
| title | Adaptable and comprehensive approaches for long-read nanopore sequencing of polyadenylated and non-polyadenylated RNAs |
| title_full | Adaptable and comprehensive approaches for long-read nanopore sequencing of polyadenylated and non-polyadenylated RNAs |
| title_fullStr | Adaptable and comprehensive approaches for long-read nanopore sequencing of polyadenylated and non-polyadenylated RNAs |
| title_full_unstemmed | Adaptable and comprehensive approaches for long-read nanopore sequencing of polyadenylated and non-polyadenylated RNAs |
| title_short | Adaptable and comprehensive approaches for long-read nanopore sequencing of polyadenylated and non-polyadenylated RNAs |
| title_sort | adaptable and comprehensive approaches for long read nanopore sequencing of polyadenylated and non polyadenylated rnas |
| topic | RNA-seq Oxford Nanopore cDNA full-length polyadenylated and non-polyadenylated long-read |
| url | https://www.frontiersin.org/articles/10.3389/fgene.2024.1466338/full |
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