TITLE:
Evaluation of Ribosomal RNA Removal Protocols for Salmonella RNA-Seq Projects
AUTHORS:
Arvind A. Bhagwat, Z. Irene Ying, Allen Smith
KEYWORDS:
Ribosomal RNA Depletion; Bacterial NextGen Sequencing; Food Borne Pathogen RNA Sequencing
JOURNAL NAME:
Advances in Microbiology,
Vol.4 No.1,
January
16,
2014
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.