Combination of reverse transcription (RT) and deep sequencing has emerged as a powerful instrument for the detection of RNA modifications, a field that has seen a recent surge in activity because of its importance in gene regulation. Recent studies yielded high-resolution RT signatures of modified ribonucleotides relying on both sequence-dependent mismatch patterns and reverse transcription arrests. Common alignment viewers lack specialized functionality, such as filtering, tailored visualization, image export and differential analysis. Consequently, the community will profit from a platform seamlessly connecting detailed visual inspection of RT signatures and automated screening for modification candidates.
Researchers at the Johannes Gutenberg University Mainz have developed CoverageAnalyzer (CAn) in response to the demand for a powerful inspection tool. It is freely available for all three main operating systems. With SAM file format as standard input, CAn is an intuitive and user-friendly tool that is generally applicable to the large community of biomedical users, starting from simple visualization of RNA sequencing (RNA-Seq) data, up to sophisticated modification analysis with significance-based modification candidate calling.
Workflow for a typical CAn session
(a) Input SAM files are processed to a positional profile; (b) Sorting and filtering of data by various statistical criteria. From the depicted result table, users select sequences for visualization; (c) Visualization tab. Independent plots and differential comparison for mismatch and/or arrest parameters with marked above‐threshold sites (yellow triangle). Display of base sequences is enabled automatically depending on the horizontal plot dimension; (d) Candidate casting tab; (i) Formula editor: Specification of screening thresholds.
Conditions are combined with Boolean operators AND, OR and XOR and can be parenthesized; (ii) Control panel for serial plotting
Availability – CoverageAnalyzer setup files (incl. source code), instructions and documentation can be downloaded at: https://zenodo.org/record/164811 or https://sourceforge.net/projects/coverageanalyzer/.