title: Next-Generation Sequencing-Based RiboMethSeq  Protocol for Analysis of tRNA 2'-O-Methylation.
abstract: 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.
authors.affiliation: IMoPA UMR7365 CNRS-UL, Biopole Lorraine University, 54505 Vandoeuvre-les-Nancy, France. Florian.Pichot5@etu.univ.|Next-Generation Sequencing core facility, FR3209 BMCT CNRS-UL, Biopole Lorraine University, 54505 Vandoeuvre-les-Nancy, France. Florian.Pichot5@etu.univ.
authors.forename: Virginie
authors.initials: V
authors.lastname: Marchand
authors.affiliation: IMoPA UMR7365 CNRS-UL, Biopole Lorraine University, 54505 Vandoeuvre-les-Nancy, France. Florian.Pichot5@etu.univ.
authors.forename: Florian
authors.initials: F
authors.lastname: Pichot
authors.affiliation: Institute of Pharmacy and Biochemistry, Johannes Gutenberg University Mainz, 55128 Mainz, Germany.
authors.forename: Kathrin
authors.initials: K
authors.lastname: Thüring
authors.affiliation: IMoPA UMR7365 CNRS-UL, Biopole Lorraine University, 54505 Vandoeuvre-les-Nancy, France. Florian.Pichot5@etu.univ.
authors.forename: Lilia
authors.initials: L
authors.lastname: Ayadi
authors.affiliation: Department of Infectious Diseases, Medical Microbiology and Hygiene, Ruprecht-Karls University Heidelberg, 69120 Heidelberg, Germany.
authors.forename: Isabel
authors.initials: I
authors.lastname: Freund
authors.affiliation: Department of Infectious Diseases, Medical Microbiology and Hygiene, Ruprecht-Karls University Heidelberg, 69120 Heidelberg, Germany.
authors.forename: Alexander
authors.initials: A
authors.lastname: Dalpke
authors.affiliation: Institute of Pharmacy and Biochemistry, Johannes Gutenberg University Mainz, 55128 Mainz, Germany.
authors.forename: Mark
authors.initials: M
authors.lastname: Helm
authors.affiliation: IMoPA UMR7365 CNRS-UL, Biopole Lorraine University, 54505 Vandoeuvre-les-Nancy, France. Florian.Pichot5@etu.univ.|Next-Generation Sequencing core facility, FR3209 BMCT CNRS-UL, Biopole Lorraine University, 54505 Vandoeuvre-les-Nancy, France. Florian.Pichot5@etu.univ.
authors.forename: Yuri
authors.initials: Y
authors.lastname: Motorin
mesh_terms: D019295:Computational Biology / Q000379:methods; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D008745:Methylation; D012329:RNA, Bacterial / Q000737:chemistry; D012331:RNA, Fungal / Q000737:chemistry; D012343:RNA, Transfer / Q000737:chemistry*; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000235:genetics; D017423:Sequence Analysis, RNA / Q000379:methods*
chemical_list: D012329:RNA, Bacterial; D012331:RNA, Fungal; D012343:RNA, Transfer
keywords: tRNA;   TrmH;  2′‐O‐methylation;  RiboMethSeq;  TRM3;  deleted strain;  high‐throughput sequencing