Evaluation of Ribosomal RNA Removal Protocols for <i>Salmonella</i> RNA-Seq Projects

Abstract

Next generation sequencing is a powerful technology whose application in sequencing entire RNA populations (RNA-Seq) of food-borne pathogens will provide valuable insights. A problem unique to prokaryotic RNA-Seq is removal of ribosomal RNA. Unlike eukaryotic messenger RNA (mRNA), bacterial mRNA species are devoid of polyadenylation at the 3'-end and thus the approach of affinity enrichment of mRNA using oligo-dT probes is not an option. Among several approaches to enriching mRNA molecules, removal of ribosomal RNA (rRNA) by subtractive hybridization has been widely used. This approach is a single-step procedure for which several rRNA-depletion kits are commercially available. We evaluated three commercially available rRNA-depletion kits to determine their respective efficiencies of rRNA removal from Salmonella enterica serovar Typhimurium strain SL1344. The three protocols achieved varying degrees of rRNA depletion and resulted in 8 to 1000-fold enrichment of mRNA. rRNA removal probes from two of the three kits were unable to titrate out 23S rRNA species while removal of 16S rRNA was less efficient. The Ribo-Zero kit was most efficient in eliminating 16S, 23S and 5S ribosomal RNA species from the transcriptome of S. enterica serovar Typhimurium strain SL1344.

Comparative evaluation of rRNA depletion procedures for the improved analysis of bacterial biofilm and mixed pathogen culture transcriptomes

“…Our findings are in agreement with previous reports supporting the efficiency of Ribo-Zero rRNA depletion for other bacterial species. For instance, only the Ribo-Zero kit was capable of successfully depleting rRNA to below 1% in Salmonella enterica serovar Typhimurium strain SL1344 samples, with rRNA accounting for more than 90% of RNA-seq reads in MICROBExpress and RiboMinus samples20. When He and colleagues assessed MICROBExpress subtractive hybridization and an exonuclease treatment alone or in combination for rRNA depletion from samples derived from two synthetic polymicrobial communities, they found that even a combination of the two methods or repeated rounds of subtractive hybridization were capable of increasing non-rRNA reads to no more than 25%11.…”

“…This is especially important considering that previous reports have demonstrated low yields of non-rRNA reads (5–30%) in cDNA libraries following depletion, with the problem being exacerbated in biofilm samples18. Despite a limited number of reports demonstrating better performance of the Ribo-Zero rRNA Removal Kit relative to the MICROBExpress kit in other bacterial species1320, publication searches limited to 2016 for “ Pseudomonas aeruginosa” or “biofilms” in combination with the respective kit names reveal similar numbers of projects utilizing the two procedures. Therefore, the present work was assessed the efficiency of three commercially available subtractive hybridization-based rRNA depletion kits: Illumina Ribo-Zero rRNA Removal Kit (Bacteria), Ambion MICROBExpress™ Bacterial mRNA Enrichment Kit and the Life Technologies RiboMinus Transcriptome Isolation Kit, Bacteria.…”

Expression of eukaryotic‐like protein in the microbiome of sponges

“…; Bhagwat et al . ), but there is little information on their performance in host–symbiont systems (Moitinho‐Silva et al . ; Versluis et al .…”

“…These two methods have been extensively used with microbial isolates or enriched communities (e.g. He et al 2010;Yi et al 2011;Giannoukos et al 2012;Peano et al 2013;Bhagwat et al 2014), but there is little information on their performance in host-symbiont systems (Moitinho-Silva et al 2014;Versluis et al 2015).…”

Benzoate-TolerantEscherichia coliStrains from Experimental Evolution Lose the Gad Regulon, Multi-Drug Efflux Pumps, and Hydrogenases

“…Illumina RNA-Seq libraries were constructed for sequencing. An enrichment of messenger RNA was achieved by depleting ribosomal RNA (rRNA) by following the guidelines of the Ribo-Zero rRNA Removal Kit (Illumina)(22, 100). The RNA-Seq library was prepared via the ScriptSeq v2 RNA-Seq library preparation kit (Epicenter, WI) with a starting concentration of 15 ng rRNA depleted RNA for each library.…”