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Advantages and challenges associated with bisulfite-assisted nanopore direct RNA sequencing for modifications
Nanopore direct RNA sequencing is a technology that allows sequencing for epitranscriptomic modifications with the possibility of a quantitative assessment. In the present work, pseudouridine (Ψ) was sequenced with the nanopore before and after the pH 7 bisulfite reaction that yields stable ribose a...
Autores principales: | , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
RSC
2023
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10619145/ https://www.ncbi.nlm.nih.gov/pubmed/37920399 http://dx.doi.org/10.1039/d3cb00081h |
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author | Fleming, Aaron M. Zhu, Judy Done, Vilhelmina K. Burrows, Cynthia J. |
author_facet | Fleming, Aaron M. Zhu, Judy Done, Vilhelmina K. Burrows, Cynthia J. |
author_sort | Fleming, Aaron M. |
collection | PubMed |
description | Nanopore direct RNA sequencing is a technology that allows sequencing for epitranscriptomic modifications with the possibility of a quantitative assessment. In the present work, pseudouridine (Ψ) was sequenced with the nanopore before and after the pH 7 bisulfite reaction that yields stable ribose adducts at C1′ of Ψ. The adducted sites produced greater base call errors in the form of deletion signatures compared to Ψ. Sequencing studies on E. coli rRNA and tmRNA before and after the pH 7 bisulfite reaction demonstrated that using chemically-assisted nanopore sequencing has distinct advantages for minimization of false positives and false negatives in the data. The rRNA from E. coli has 19 known U/C sequence variations that give similar base call signatures as Ψ, and therefore, are false positives when inspecting base call data; however, these sites are refractory to reacting with bisulfite as is easily observed in nanopore data. The E. coli tmRNA has a low occupancy Ψ in a pyrimidine-rich sequence context that is called a U representing a false negative; partial occupancy by Ψ is revealed after the bisulfite reaction. In a final study, 5-methylcytidine (m(5)C) in RNA can readily be observed after the pH 5 bisulfite reaction in which the parent C deaminates to U and the modified site does not react. This locates m(5)C when using bisulfite-assisted nanopore direct RNA sequencing, which is otherwise challenging to observe. The advantages and challenges of the overall approach are discussed. |
format | Online Article Text |
id | pubmed-10619145 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | RSC |
record_format | MEDLINE/PubMed |
spelling | pubmed-106191452023-11-02 Advantages and challenges associated with bisulfite-assisted nanopore direct RNA sequencing for modifications Fleming, Aaron M. Zhu, Judy Done, Vilhelmina K. Burrows, Cynthia J. RSC Chem Biol Chemistry Nanopore direct RNA sequencing is a technology that allows sequencing for epitranscriptomic modifications with the possibility of a quantitative assessment. In the present work, pseudouridine (Ψ) was sequenced with the nanopore before and after the pH 7 bisulfite reaction that yields stable ribose adducts at C1′ of Ψ. The adducted sites produced greater base call errors in the form of deletion signatures compared to Ψ. Sequencing studies on E. coli rRNA and tmRNA before and after the pH 7 bisulfite reaction demonstrated that using chemically-assisted nanopore sequencing has distinct advantages for minimization of false positives and false negatives in the data. The rRNA from E. coli has 19 known U/C sequence variations that give similar base call signatures as Ψ, and therefore, are false positives when inspecting base call data; however, these sites are refractory to reacting with bisulfite as is easily observed in nanopore data. The E. coli tmRNA has a low occupancy Ψ in a pyrimidine-rich sequence context that is called a U representing a false negative; partial occupancy by Ψ is revealed after the bisulfite reaction. In a final study, 5-methylcytidine (m(5)C) in RNA can readily be observed after the pH 5 bisulfite reaction in which the parent C deaminates to U and the modified site does not react. This locates m(5)C when using bisulfite-assisted nanopore direct RNA sequencing, which is otherwise challenging to observe. The advantages and challenges of the overall approach are discussed. RSC 2023-08-23 /pmc/articles/PMC10619145/ /pubmed/37920399 http://dx.doi.org/10.1039/d3cb00081h Text en This journal is © The Royal Society of Chemistry https://creativecommons.org/licenses/by-nc/3.0/ |
spellingShingle | Chemistry Fleming, Aaron M. Zhu, Judy Done, Vilhelmina K. Burrows, Cynthia J. Advantages and challenges associated with bisulfite-assisted nanopore direct RNA sequencing for modifications |
title | Advantages and challenges associated with bisulfite-assisted nanopore direct RNA sequencing for modifications |
title_full | Advantages and challenges associated with bisulfite-assisted nanopore direct RNA sequencing for modifications |
title_fullStr | Advantages and challenges associated with bisulfite-assisted nanopore direct RNA sequencing for modifications |
title_full_unstemmed | Advantages and challenges associated with bisulfite-assisted nanopore direct RNA sequencing for modifications |
title_short | Advantages and challenges associated with bisulfite-assisted nanopore direct RNA sequencing for modifications |
title_sort | advantages and challenges associated with bisulfite-assisted nanopore direct rna sequencing for modifications |
topic | Chemistry |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10619145/ https://www.ncbi.nlm.nih.gov/pubmed/37920399 http://dx.doi.org/10.1039/d3cb00081h |
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