title: RoboOligo: software for mass spectrometry data to support manual and de novo sequencing of post-transcriptionally modified ribonucleic acids.
abstract: Ribosomal ribonucleic acid (RNA), transfer RNA and other biological or synthetic RNA polymers can contain nucleotides that have been modified by the addition of chemical groups. Traditional Sanger sequencing methods cannot establish the chemical nature and sequence of these modified-nucleotide containing oligomers. Mass spectrometry (MS) has become the conventional approach for determining the nucleotide composition, modification status and sequence of modified RNAs. Modified RNAs are analyzed by MS using collision-induced dissociation tandem mass spectrometry (CID MS/MS), which produces a complex dataset of oligomeric fragments that must be interpreted to identify and place modified nucleosides within the RNA sequence. Here we report the development of RoboOligo, an interactive software program for the robust analysis of data generated by CID MS/MS of RNA oligomers. There are three main functions of RoboOligo: (i) automated de novo sequencing via the local search paradigm. (ii) Manual sequencing with real-time spectrum labeling and cumulative intensity scoring. (iii) A hybrid approach, coined 'variable sequencing', which combines the user intuition of manual sequencing with the high-throughput sampling of automated de novo sequencing.
authors.affiliation: Department of Microbiology and The Center for RNA Biology, The Ohio State University, Columbus, OH 43210, USA Pat.Limbach@uc.edu.
authors.forename: Paul J
authors.initials: PJ
authors.lastname: Sample
authors.affiliation: Rieveschl Laboratories for Mass Spectrometry, Department of Chemistry, PO Box 210172, University of Cincinnati, Cincinnati, OH 45221-0172, USA.
authors.forename: Kirk W
authors.initials: KW
authors.lastname: Gaston
authors.affiliation: Department of Microbiology and The Center for RNA Biology, The Ohio State University, Columbus, OH 43210, USA Ohio State Biochemistry Program, The Ohio State University, Columbus, OH 43210, USA.
authors.forename: Juan D
authors.initials: JD
authors.lastname: Alfonzo
authors.affiliation: Rieveschl Laboratories for Mass Spectrometry, Department of Chemistry, PO Box 210172, University of Cincinnati, Cincinnati, OH 45221-0172, USA Pat.Limbach@uc.edu.
authors.forename: Patrick A
authors.initials: PA
authors.lastname: Limbach
mesh_terms: D000465:Algorithms; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D012323:RNA Processing, Post-Transcriptional; D012335:RNA, Ribosomal / Q000737:chemistry / Q000378:metabolism; D012343:RNA, Transfer / Q000737:chemistry / Q000378:metabolism; D017423:Sequence Analysis, RNA / Q000379:methods*; D012984:Software; D053719:Tandem Mass Spectrometry
chemical_list: D012335:RNA, Ribosomal; D012343:RNA, Transfer
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