Next‐Generation Sequencing‐Based RiboMethSeq Protocol for Analysis of tRNA 2′‐O‐Methylation
Analysis of RNA modifications by traditional physico‐chemical approaches is labor intensive, requires substantial amounts of input material and only allows site‐by‐site measurements. The recent development of qualitative and quantitative approaches based on next‐generation se...
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doaj-edf64eb77b2143c28ca5e3ae3724d0a02020-11-24T21:58:53ZengMDPI AGBiomolecules2218-273X2017-02-01711310.3390/biom7010013biom7010013Next‐Generation Sequencing‐Based RiboMethSeq Protocol for Analysis of tRNA 2′‐O‐MethylationVirginie Marchand0Florian Pichot1Kathrin Thüring2Lilia Ayadi3Isabel Freund4Alexander Dalpke5Mark Helm6Yuri Motorin7IMoPA UMR7365 CNRS‐UL, Biopole Lorraine University, 54505 Vandoeuvre‐les‐Nancy, FranceIMoPA UMR7365 CNRS‐UL, Biopole Lorraine University, 54505 Vandoeuvre‐les‐Nancy, FranceInstitute of Pharmacy and Biochemistry, Johannes Gutenberg University Mainz, 55128 Mainz, GermanyIMoPA UMR7365 CNRS‐UL, Biopole Lorraine University, 54505 Vandoeuvre‐les‐Nancy, FranceDepartment of Infectious Diseases, Medical Microbiology and Hygiene, Ruprecht‐Karls University Heidelberg, 69120 Heidelberg, GermanyDepartment of Infectious Diseases, Medical Microbiology and Hygiene, Ruprecht‐Karls University Heidelberg, 69120 Heidelberg, GermanyInstitute of Pharmacy and Biochemistry, Johannes Gutenberg University Mainz, 55128 Mainz, GermanyIMoPA UMR7365 CNRS‐UL, Biopole Lorraine University, 54505 Vandoeuvre‐les‐Nancy, FranceAnalysis of RNA modifications by traditional physico‐chemical approaches is labor intensive, requires substantial amounts of input material and only allows site‐by‐site measurements. The recent development of qualitative and quantitative approaches based on next‐generation sequencing (NGS) opens new perspectives for the analysis of various cellular RNA species. The Illumina sequencing‐based RiboMethSeq protocol was initially developed and successfully applied for mapping of ribosomal RNA (rRNA) 2′‐O‐methylations. This method also gives excellent results in the quantitative analysis of rRNA modifications in different species and under varying growth conditions. However, until now, RiboMethSeq was only employed for rRNA, and the whole sequencing and analysis pipeline was only adapted to this long and rather conserved RNA species. A deep understanding of RNA modification functions requires large and global analysis datasets for other important RNA species, namely for transfer RNAs (tRNAs), which are well known to contain a great variety of functionally‐important modified residues. Here, we evaluated the application of the RiboMethSeq protocol for the analysis of tRNA 2′‐O‐methylation in Escherichia coli and in Saccharomyces cerevisiae. After a careful optimization of the bioinformatic pipeline, RiboMethSeq proved to be suitable for relative quantification of methylation rates for known modified positions in different tRNA species.http://www.mdpi.com/2218-273X/7/1/13tRNA 2′‐O‐methylation RiboMethSeq high‐throughput sequencing deleted strain TrmH TRM3 |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Virginie Marchand Florian Pichot Kathrin Thüring Lilia Ayadi Isabel Freund Alexander Dalpke Mark Helm Yuri Motorin |
spellingShingle |
Virginie Marchand Florian Pichot Kathrin Thüring Lilia Ayadi Isabel Freund Alexander Dalpke Mark Helm Yuri Motorin Next‐Generation Sequencing‐Based RiboMethSeq Protocol for Analysis of tRNA 2′‐O‐Methylation Biomolecules tRNA 2′‐O‐methylation RiboMethSeq high‐throughput sequencing deleted strain TrmH TRM3 |
author_facet |
Virginie Marchand Florian Pichot Kathrin Thüring Lilia Ayadi Isabel Freund Alexander Dalpke Mark Helm Yuri Motorin |
author_sort |
Virginie Marchand |
title |
Next‐Generation Sequencing‐Based RiboMethSeq Protocol for Analysis of tRNA 2′‐O‐Methylation |
title_short |
Next‐Generation Sequencing‐Based RiboMethSeq Protocol for Analysis of tRNA 2′‐O‐Methylation |
title_full |
Next‐Generation Sequencing‐Based RiboMethSeq Protocol for Analysis of tRNA 2′‐O‐Methylation |
title_fullStr |
Next‐Generation Sequencing‐Based RiboMethSeq Protocol for Analysis of tRNA 2′‐O‐Methylation |
title_full_unstemmed |
Next‐Generation Sequencing‐Based RiboMethSeq Protocol for Analysis of tRNA 2′‐O‐Methylation |
title_sort |
next‐generation sequencing‐based ribomethseq protocol for analysis of trna 2′‐o‐methylation |
publisher |
MDPI AG |
series |
Biomolecules |
issn |
2218-273X |
publishDate |
2017-02-01 |
description |
Analysis of RNA modifications by traditional physico‐chemical approaches is labor intensive, requires substantial amounts of input material and only allows site‐by‐site measurements. The recent development of qualitative and quantitative approaches based on next‐generation sequencing (NGS) opens new perspectives for the analysis of various cellular RNA species. The Illumina sequencing‐based RiboMethSeq protocol was initially developed and successfully applied for mapping of ribosomal RNA (rRNA) 2′‐O‐methylations. This method also gives excellent results in the quantitative analysis of rRNA modifications in different species and under varying growth conditions. However, until now, RiboMethSeq was only employed for rRNA, and the whole sequencing and analysis pipeline was only adapted to this long and rather conserved RNA species. A deep understanding of RNA modification functions requires large and global analysis datasets for other important RNA species, namely for transfer RNAs (tRNAs), which are well known to contain a great variety of functionally‐important modified residues. Here, we evaluated the application of the RiboMethSeq protocol for the analysis of tRNA 2′‐O‐methylation in Escherichia coli and in Saccharomyces cerevisiae. After a careful optimization of the bioinformatic pipeline, RiboMethSeq proved to be suitable for relative quantification of methylation rates for known modified positions in different tRNA species. |
topic |
tRNA 2′‐O‐methylation RiboMethSeq high‐throughput sequencing deleted strain TrmH TRM3 |
url |
http://www.mdpi.com/2218-273X/7/1/13 |
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