Computational protocol: Cartography of Methicillin-Resistant S. aureus Transcripts: Detection, Orientation and Temporal Expression during Growth Phase and Stress Conditions

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Protocol publication

[…] All the small RNA transcripts detected in intergenic regions by the mapping of all transcripts in the Artemis annotation environment were analyzed with regard to their location, their characteristics, their potential function and their previous identification in the literature. For each intergenic small RNA transcript, several analyses were performed. We identified the presence of a putative related ribosome binding site and/or a promoter at the 5′-end. We checked the 3′-end for the presence of a rho-independent terminator of transcription. The orientation of the neighbor genes was also considered to classify small RNA transcripts. Five classes of small RNA transcripts were therefore defined that included: putative small CDSs, cis-acting regulatory elements, riboswitches, bona fide small RNAs and antisense RNAs. We computed the GC content of each small RNA transcript, considering that the presence of a bias in the GC content would be an argument in favor of bona fide small RNA as it is the case for tRNA and rRNA in that species. The presence of such a bias has been evaluated by considering two standard deviations of the lower tRNA GC percentage in comparison with the average GC percentage of the S. aureus N315 genome (32.5%). The cut-off for such a higher GC percentage was set to 42%. For each category, we scanned the transcript for the presence of a UCCC motif as described previously . All small RNA transcripts were also compared to those identified by the five previously published studies , , –. In that case, only the locus was considered for all overlapping identified regions (not the strand orientation). Data available in the study of Anderson and colleagues were mapped to the N315 genomic sequence by using the Basic Local Alignment Tool with default parameters. The sequence of each small RNA transcript was enriched by annotation data whenever possible by considering both RFAM and Genbank databanks and the literature for sprA–G sRNAs and other stable rsa RNAs, . NCBI and RFAM databases provided more than 110 annotated RNAs for the S. aureus N315 genome including essentially tRNAs and rRNAs at NCBI while other putative sRNAs and riboswitches were found annotated in the RFAM database. For all the transcripts that lacked an annotation, a comparative analysis was performed by using BLAST with default parameters. This analysis was completed by both the HMM approach previously described by Geissmann to identify GC-rich regions and RNasim comparative analysis that included Wu-blast 2.0 pairwise comparisons of sequences for searching similarities, and QRNA to identify base substitution patterns in pairwise alignments that could correspond to a conserved RNA secondary structure. The interpretation of our data was facilitated by the ApolloRNA program, which was used to assign and visualize all the available information on the annotated S. aureus N315 genome . Structure prediction was performed as previously described . […]

Pipeline specifications

Software tools WU-BLAST, QRNA
Databases Rfam
Applications RNA structure analysis, Transcription analysis
Organisms Staphylococcus aureus, Epipremnum aureum, Staphylococcus aureus subsp. aureus N315
Diseases Infection
Chemicals Methicillin