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Semi-quantitative detection of pseudouridine modifications and type I/II hypermodifications in human mRNAs using direct long-read sequencing
Here, we develop and apply a semi-quantitative method for the high-confidence identification of pseudouridylated sites on mammalian mRNAs via direct long-read nanopore sequencing. A comparative analysis of a modification-free transcriptome reveals that the depth of coverage and specific k-mer sequen...
Autores principales: | , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Nature Publishing Group UK
2023
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9852470/ https://www.ncbi.nlm.nih.gov/pubmed/36658122 http://dx.doi.org/10.1038/s41467-023-35858-w |
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author | Tavakoli, Sepideh Nabizadeh, Mohammad Makhamreh, Amr Gamper, Howard McCormick, Caroline A. Rezapour, Neda K. Hou, Ya-Ming Wanunu, Meni Rouhanifard, Sara H. |
author_facet | Tavakoli, Sepideh Nabizadeh, Mohammad Makhamreh, Amr Gamper, Howard McCormick, Caroline A. Rezapour, Neda K. Hou, Ya-Ming Wanunu, Meni Rouhanifard, Sara H. |
author_sort | Tavakoli, Sepideh |
collection | PubMed |
description | Here, we develop and apply a semi-quantitative method for the high-confidence identification of pseudouridylated sites on mammalian mRNAs via direct long-read nanopore sequencing. A comparative analysis of a modification-free transcriptome reveals that the depth of coverage and specific k-mer sequences are critical parameters for accurate basecalling. By adjusting these parameters for high-confidence U-to-C basecalling errors, we identify many known sites of pseudouridylation and uncover previously unreported uridine-modified sites, many of which fall in k-mers that are known targets of pseudouridine synthases. Identified sites are validated using 1000-mer synthetic RNA controls bearing a single pseudouridine in the center position, demonstrating systematic under-calling using our approach. We identify mRNAs with up to 7 unique modification sites. Our workflow allows direct detection of low-, medium-, and high-occupancy pseudouridine modifications on native RNA molecules from nanopore sequencing data and multiple modifications on the same strand. |
format | Online Article Text |
id | pubmed-9852470 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | Nature Publishing Group UK |
record_format | MEDLINE/PubMed |
spelling | pubmed-98524702023-01-21 Semi-quantitative detection of pseudouridine modifications and type I/II hypermodifications in human mRNAs using direct long-read sequencing Tavakoli, Sepideh Nabizadeh, Mohammad Makhamreh, Amr Gamper, Howard McCormick, Caroline A. Rezapour, Neda K. Hou, Ya-Ming Wanunu, Meni Rouhanifard, Sara H. Nat Commun Article Here, we develop and apply a semi-quantitative method for the high-confidence identification of pseudouridylated sites on mammalian mRNAs via direct long-read nanopore sequencing. A comparative analysis of a modification-free transcriptome reveals that the depth of coverage and specific k-mer sequences are critical parameters for accurate basecalling. By adjusting these parameters for high-confidence U-to-C basecalling errors, we identify many known sites of pseudouridylation and uncover previously unreported uridine-modified sites, many of which fall in k-mers that are known targets of pseudouridine synthases. Identified sites are validated using 1000-mer synthetic RNA controls bearing a single pseudouridine in the center position, demonstrating systematic under-calling using our approach. We identify mRNAs with up to 7 unique modification sites. Our workflow allows direct detection of low-, medium-, and high-occupancy pseudouridine modifications on native RNA molecules from nanopore sequencing data and multiple modifications on the same strand. Nature Publishing Group UK 2023-01-19 /pmc/articles/PMC9852470/ /pubmed/36658122 http://dx.doi.org/10.1038/s41467-023-35858-w Text en © The Author(s) 2023 https://creativecommons.org/licenses/by/4.0/Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) . |
spellingShingle | Article Tavakoli, Sepideh Nabizadeh, Mohammad Makhamreh, Amr Gamper, Howard McCormick, Caroline A. Rezapour, Neda K. Hou, Ya-Ming Wanunu, Meni Rouhanifard, Sara H. Semi-quantitative detection of pseudouridine modifications and type I/II hypermodifications in human mRNAs using direct long-read sequencing |
title | Semi-quantitative detection of pseudouridine modifications and type I/II hypermodifications in human mRNAs using direct long-read sequencing |
title_full | Semi-quantitative detection of pseudouridine modifications and type I/II hypermodifications in human mRNAs using direct long-read sequencing |
title_fullStr | Semi-quantitative detection of pseudouridine modifications and type I/II hypermodifications in human mRNAs using direct long-read sequencing |
title_full_unstemmed | Semi-quantitative detection of pseudouridine modifications and type I/II hypermodifications in human mRNAs using direct long-read sequencing |
title_short | Semi-quantitative detection of pseudouridine modifications and type I/II hypermodifications in human mRNAs using direct long-read sequencing |
title_sort | semi-quantitative detection of pseudouridine modifications and type i/ii hypermodifications in human mrnas using direct long-read sequencing |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9852470/ https://www.ncbi.nlm.nih.gov/pubmed/36658122 http://dx.doi.org/10.1038/s41467-023-35858-w |
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