ShapeFinder: A software system for high-throughput quantitative analysis of nucleic acid reactivity information resolved by capillary electrophoresis
- 1Department of Biomedical Engineering, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA
- 2Swiss Institute of Bioinformatics, CH-1015 Lausanne, Switzerland
- 3Department of Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA
- 4Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA
- 5Department of Computer Science, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA
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↵6 These authors contributed equally to this work.
Abstract
Analysis of the long-range architecture of RNA is a challenging experimental and computational problem. Local nucleotide flexibility, which directly reports underlying base pairing and tertiary interactions in an RNA, can be comprehensively assessed at single nucleotide resolution using high-throughput selective 2′-hydroxyl acylation analyzed by primer extension (hSHAPE). hSHAPE resolves structure-sensitive chemical modification information by high-resolution capillary electrophoresis and typically yields quantitative nucleotide flexibility information for 300–650 nucleotides (nt) per experiment. The electropherograms generated in hSHAPE experiments provide a wealth of structural information; however, significant algorithmic analysis steps are required to generate quantitative and interpretable data. We have developed a set of software tools called ShapeFinder to make possible rapid analysis of raw sequencer data from hSHAPE, and most other classes of nucleic acid reactivity experiments. The algorithms in ShapeFinder (1) convert measured fluorescence intensity to quantitative cDNA fragment amounts, (2) correct for signal decay over read lengths extending to 600 nts or more, (3) align reactivity data to the known RNA sequence, and (4) quantify per nucleotide reactivities using whole-channel Gaussian integration. The algorithms and user interface tools implemented in ShapeFinder create new opportunities for tackling ambitious problems involving high-throughput analysis of structure–function relationships in large RNAs.
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Footnotes
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Reprint requests to: Kevin M. Weeks, Department of Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA; e-mail: weeks{at}unc.edu; fax: (919) 962-2388; or Morgan C. Giddings, Department of Biomedical Engineering, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA; e-mail: giddings{at}unc.edu; fax: (919) 962-2388.
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Article published online ahead of print. Article and publication date are at http://www.rnajournal.org/cgi/doi/10.1261/rna.1166808.
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- Received May 2, 2008.
- Accepted July 9, 2008.
- Copyright © 2008 RNA Society
