REDIportal: millions of novel A-to-I RNA editing events from thousands of RNAseq experiments

Abstract

RNA editing is a relevant epitranscriptome phenomenon able to increase the transcriptome and proteome diversity of eukaryotic organisms. ADAR mediated RNA editing is widespread in humans in which millions of A-to-I changes modify thousands of primary transcripts. RNA editing has pivotal roles in the regulation of gene expression or modulation of the innate immune response or functioning of several neurotransmitter receptors. Massive transcriptome sequencing has fostered the research in this field. Nonetheless, different aspects of the RNA editing biology are still unknown and need to be elucidated. To support the study of A-to-I RNA editing we have updated our REDIportal catalogue raising its content to about 16 millions of events detected in 9642 human RNAseq samples from the GTEx project by using a dedicated pipeline based on the HPC version of the REDItools software. REDIportal now allows searches at sample level, provides overviews of RNA editing profiles per each RNAseq experiment, implements a Gene View module to look at individual events in their genic context and hosts the CLAIRE database. Starting from this novel version, REDIportal will start collecting non-human RNA editing changes for comparative genomics investigations. The database is freely available at http://srv00.recas.ba.infn.it/atlas/index.html.

Figure 1.

Data processing and database construction. (A) RNAseq data in fastq format are aligned on the human genome by STAR and converted in BAM files. In parallel, if DNAseq reads are available, are aligned on the same genome version by BWA. RNAseq BAM files are analyzed by HPC REDItools and the editing calling is distribute across different computing nodes, each working on a given genomic region. Resulting REDItools tables undergo to further filtering steps before the generation of the final table of A-to-I candidates. RNAseq unmapped reads are re-analyzed to detect hyper-edited reads and provide a list of hyper-editing sites per sample. RNAseq BAM files are further used to calculate the AEI and REI indices. (B) REDItools table and hyper-editing tables are merged in the final REDIportal collection. All events are annotated and stored in the MySQL TABLE1. They are also used to interrogate all RNAseq data to recover RNA editing levels and populate the MySQL TABLE2. Main RNA editing statistics per sample are also computed and collected in the MySQL TABLE3. Blue rectangles are reads, red rectangles are genomic regions, while black stars are A-to-I candidates.

Figure 2.

Example of Gene View page for gene MRI1. Gene View is a novel REDItools functionality in which users can visualize RNA editing events in their genic context. The web page shows at most three panels: (i) the gene structure with details about individual transcripts and specific features such as 5UTR for 5′ UTR in blue, 3UTR for 3′ UTR in red, CDS for the protein coding region in orange, Intron for intervening sequences in black and Exon for a non-coding exon in green; (ii) the list of RNA editing events in blue circles with related levels (the mean value is included in case of searches from the Gene View search page); (iii) the RNAseq coverage for the specific genomic region and related to the specific RNAseq experiment. This panel is visible only at sample level.

Figure 3.

Example of sample search for run SRR1069188. The output of the sample search provides basic RNA editing info per RNAseq in a tabular format and enables the browsing of five panels with further details. (A) ‘Genomics Facts’ includes the location and distribution of detected sites in different genomic and genic regions; (B) ‘Base Distribution’ shows the graphical distribution of all detected variants by the REDItools based pipeline; (C) ‘RNA Editing Indices’ depicts box plots of AEI and REI indices calculated on the body site group of the retrieved sample. Specific index values are indicated in each plot by an empty circle; (D) ‘RNA Editing Levels’ shows the distribution of RNA editing levels; (E) ‘Transcriptome Coverage’ displays statistics about the fraction of edited and unedited genes, and the distribution of detected events in mRNAs or ncRNAs. Panels C and E include two additional buttons for further details about recoding events (Figure 3) and edited genes.

Figure 4.

Details about recoding events. The ‘RNA Editing Indices’ panel comprises a button enabling the browsing of individual recoding events. Selected positions can be compared by a bar graph or all sites can be shown in dedicated plots.