title: High-throughput sequencing for 1-methyladenosine (m(1)A) mapping in RNA.
abstract: Detection and mapping of modified nucleotides in RNAs is a difficult and laborious task. Several physico-chemical approaches based on differential properties of modified nucleotides can be used, however, most of these methods do not allow high-throughput analysis. Here we describe in details a method for mapping of rather common 1-methyladenosine (m(1)A) residues using high-throughput next generation sequencing (NGS). Since m(1)A residues block primer extension during reverse transcription (RT), the accumulation of abortive products as well as the nucleotide misincorporation can be detected in the sequencing data. The described library preparation protocol allows to capture both types of cDNA products essential for further bioinformatic analysis. We demonstrate that m(1)A residues produce characteristic arrest and mismatch rates and combination of both can be used for their detection as well as for discrimination of m(1)A from other modified A residues present in RNAs.
authors.affiliation: Institute of Pharmacy and Biochemistry, Johannes Gutenberg University Mainz, Staudingerweg 5, 55128 Mainz, Germany.
authors.forename: Lyudmil
authors.initials: L
authors.lastname: Tserovski
authors.affiliation: IMoPA UMR7365 CNRS-UL, BioPole Lorraine University, 9 avenue de la Foret de Haye, 54505 Vandoeuvre-les-Nancy, France; Next-Generation Sequencing Core Facility, FR3209 BMCT, Lorraine University, 9 avenue de la Foret de Haye, 54505 Vandoeuvre-les-Nancy, France.
authors.forename: Virginie
authors.initials: V
authors.lastname: Marchand
authors.affiliation: Institute of Pharmacy and Biochemistry, Johannes Gutenberg University Mainz, Staudingerweg 5, 55128 Mainz, Germany.
authors.forename: Ralf
authors.initials: R
authors.lastname: Hauenschild
authors.affiliation: IMoPA UMR7365 CNRS-UL, BioPole Lorraine University, 9 avenue de la Foret de Haye, 54505 Vandoeuvre-les-Nancy, France; Next-Generation Sequencing Core Facility, FR3209 BMCT, Lorraine University, 9 avenue de la Foret de Haye, 54505 Vandoeuvre-les-Nancy, France.
authors.forename: Florence
authors.initials: F
authors.lastname: Blanloeil-Oillo
authors.affiliation: Institute of Pharmacy and Biochemistry, Johannes Gutenberg University Mainz, Staudingerweg 5, 55128 Mainz, Germany.
authors.forename: Mark
authors.initials: M
authors.lastname: Helm
authors.affiliation: IMoPA UMR7365 CNRS-UL, BioPole Lorraine University, 9 avenue de la Foret de Haye, 54505 Vandoeuvre-les-Nancy, France; Next-Generation Sequencing Core Facility, FR3209 BMCT, Lorraine University, 9 avenue de la Foret de Haye, 54505 Vandoeuvre-les-Nancy, France. Electronic address: Yuri.Motorin@univ-lorraine.fr.
authors.forename: Yuri
authors.initials: Y
authors.lastname: Motorin
mesh_terms: D000241:Adenosine / Q000031:analogs & derivatives* / Q000235:genetics / Q000302:isolation & purification; D015723:Gene Library; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D012313:RNA / Q000235:genetics*; D012323:RNA Processing, Post-Transcriptional / Q000235:genetics*
chemical_list: C002230:1-methyladenosine; D012313:RNA; D000241:Adenosine
keywords: High-throughput sequencing; Misincorporation signature; RNA modification; Reverse transcription