Computational protocol: RNA Editing During Sexual Development Occurs in Distantly Related Filamentous Ascomycetes

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Protocol publication

[…] Publicly available RNA-seq data () were analyzed for the presence of putative editing sites. First, raw sequence reads from S. macrospora and P. confluens were trimmed to remove undetermined bases and polyA/polyT stretches from the ends, and quality trimming from the 3' and 5' end was performed until the base quality score was at least 10. Trimmed reads of at least 40 bases were mapped onto the predicted gene sequences (coding sequences and untranslated regions including introns) based on the genome annotation of S. macrospora and P. confluens (, , ) using Tophat version 2.1.1 (). The shorter reads of S. pombe () were mapped directly without trimming. Reads were mapped onto gene sequences (including coding sequences, introns, and untranslated regions) instead of genome sequences to be able to directly identify A-to-G changes (when mapping onto genome sequences, A-to-G changes in genes encoded on the reverse strand would appear as T-to-C instead). Based on the mapped reads, the mpileup function of SAMtools () was used to generate coverage information for each base in the predicted RNAs for each of the analyzed samples. Custom-made Perl scripts were used to identify putative sequence variants from the coverage information. Variants were filtered for putative editing sites using custom-made Perl scripts that retained only variants with a single alternative base (i.e. no insertions/deletions or positions with more than one base difference from the reference genome), a minimal coverage of five reads (S. macrospora, P. confluens) or three reads (S. pombe, the lower threshold was used due to lower read coverage), at least 3% and two reads coverage of the alternative base, similar to the conditions used in a previous study for F. graminearum (). It was then analyzed which variants were present in both independent biological replicates available for each analyzed condition (), and only these reproducibly identified variants were analyzed further using custom-made Perl scripts.The strain used for the RNA-seq analysis was identical to the one used for genome sequencing in the case of P. confluens (). For S. macrospora, the wild type strain used for RNA-seq was a derivative of the wild type strain used for the original genome sequencing (); however the genome version (v02) used in our analysis is based on corrections of the original genome sequence based on resequencing of the strain also used for RNA-seq (, ). Therefore, strain differences should not contribute significantly to differences between the RNA-seq data and the genome sequences of the two species. However, to check to what degree such differences or errors in the reference genome sequences contributed to the identified putative editing sites, we analyzed how many putative editing sites occurred in the RNA-seq data from all samples with at least 95% of the variant base. This resulted in 15 sites for S. macrospora and 13 sites for P. confluens. Thus, the large majority of the hundreds to thousands of potential editing sites that were identified (see below) are not due to strain differences or errors in the genome sequences.For a comparison of editing sites of S. macrospora with P. confluens and F. graminearum, respectively, orthologs between S. macrospora and the other two fungi were identified by reciprocal BLAST as described (, ). Putative editing sites identified in wild type protoperithecial samples from S. macrospora were compared with F. graminearum editing sites found by and with putative editing sites identified in sexual tissue from P. confluens. Functional classification of genes with putative editing sites was done using FungiFun 2 () with the FunCat ontology (), or Ontologizer () based on gene ontology (GO) annotations from UniProt (, ). […]

Pipeline specifications

Software tools TopHat, SAMtools, FungiFun, Ontologizer
Databases FunCat
Application RNA-seq analysis
Organisms Fusarium graminearum, Sordaria macrospora, Pyronema omphalodes, Schizosaccharomyces pombe