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

Abstract

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.

Improved bacterial RNA-seq by Cas9-based depletion of ribosomal RNA reads

“…Available alternative bacterial rRNA removal strategies use RNase H-mediated digestion of RNA:DNA hybrids (Huang et al 2020), size-selection of mRNAs through liquid chromatography (Castro et al 2013), or selective exclusion of rRNAs from cDNA conversion with "not-so-random" hexamers (Hirakawa et al 2011;Chugani et al 2012); the latter is also available as part of the Universal Prokaryotic RNA-Seq kit (NuGen). Overall, these methods achieve good to excellent (∼70%-99%) rRNA depletion rates.…”

Differential gene expression profile of Shigella dysenteriae causing bacteremia in an immunocompromised individual

“…The invasive traits of selected isolates were studied by comparing the differential gene expression profile of the strain isolated exclusively from stool and blood specimen concurrently by transcriptomics. RNA-sequencing procedure was performed according to the manufacturer's instructions using Ion Torrent (PGM) sequencer with 400-bp read chemistry (Life Technologies, CA, USA) [10]. The quality and quantity of each library was determined at each step with a Qubit R 3.0 Fluorometer.…”

Progression of ‘Omics’ Methodologies for Understanding the Pathogenicity of Corynebacterium Pseudotuberculosis: The Brazilian Experience

“…Because large portions of sequencing reads are mapped to ribosomal RNA genes, Castro and colleagues [41] tested a new methodology, based on denaturing high-performance liquid chromatography, to deplete ribosomal transcripts from bacterial total RNA samples using C. pseudotuberculosis (biovar equi ) as a model organism. With the elimination of 78% to 92% of rRNA, which are levels that resemble those obtained with a conventional subtraction kit, this new method offers financial advantages for researchers who have access to a chromatographic system.…”

“…The assessment of global transcriptional profiles in bacteria constitutes a key strategy for unveiling mechanisms that are important for virulence and pathogenicity; thus, any efforts to increase the feasibility of RNA-seq experiments are welcome when studying pathogens such as C. pseudotuberculosis . Because large portions of sequencing reads are mapped to ribosomal RNA genes, Castro and colleagues [ 41 ] tested a new methodology, based on denaturing high-performance liquid chromatography, to deplete ribosomal transcripts from bacterial total RNA samples using C. pseudotuberculosis (biovar equi ) as a model organism. With the elimination of 78% to 92% of rRNA, which are levels that resemble those obtained with a conventional subtraction kit, this new method offers financial advantages for researchers who have access to a chromatographic system.…”