Ion Torrent‐based transcriptional assessment of a Corynebacterium pseudotuberculosis equi strain reveals denaturing high‐performance liquid chromatography a promising rRNA depletion method

Summary Corynebacterium pseudotuberculosis equi is a Gram‐positive pathogenic bacterium which affects a variety of hosts. Besides the great economic losses it causes to horse‐breeders, this organism is also known to be an important infectious agent to cattle and buffaloes. As an outcome of the efforts in characterizing the molecular basis of its virulence, several complete genome sequences were made available in recent years, enabling the large‐scale assessment of genes throughout distinct isolates. Meanwhile, the RNA‐seq stood out as the technology of choice for comprehensive transcriptome studies, which may bring valuable information regarding active genomic regions, despite of the still impeditive associated costs. In an attempt to increase the use of generated reads per instrument run, by effectively eliminating unwanted rRNAs from total RNA samples without relying on any commercially available kits, we applied denaturing high‐performance liquid chromatography (DHPLC) as an alternative method to assess the transcriptional profile of C. pseudotuberculosis. We have found that the DHPLC depletion method, allied to Ion Torrent sequencing, allows mapping of transcripts in a comprehensive way and identifying novel transcripts when a de novo approach is used. These data encourage us to use DHPLC in future transcriptional evaluations in C. pseudotuberculosis. We hereby demonstrate an alternative rRNA depletion method which was tested in order to assess the transcriptional activity of a Corynebacterium pseudotuberculosis biovar equi strain using the Ion Torrent sequencing technology. This method, based on denaturing high‐performance liquid chromatography (DHPLC), showed to subtract ribosomal transcripts in levels which resemble those seen when a commercial depletion kit was used. The DHPLC‐based depletion method could be possibly employed to treat total RNA of other organisms and represent a cost‐effective option when a suitable chromatographic system is available.