title: Comparative evaluation of rRNA depletion procedures for the improved analysis of bacterial biofilm and mixed pathogen culture transcriptomes.
abstract: Global transcriptomic analysis via RNA-seq is often hampered by the high abundance of ribosomal (r)RNA in bacterial cells. To remove rRNA and enrich coding sequences, subtractive hybridization procedures have become the approach of choice prior to RNA-seq, with their efficiency varying in a manner dependent on sample type and composition. Yet, despite an increasing number of RNA-seq studies, comparative evaluation of bacterial rRNA depletion methods has remained limited. Moreover, no such study has utilized RNA derived from bacterial biofilms, which have potentially higher rRNA:mRNA ratios and higher rRNA carryover during RNA-seq analysis. Presently, we evaluated the efficiency of three subtractive hybridization-based kits in depleting rRNA from samples derived from biofilm, as well as planktonic cells of the opportunistic human pathogen Pseudomonas aeruginosa. Our results indicated different rRNA removal efficiency for the three procedures, with the Ribo-Zero kit yielding the highest degree of rRNA depletion, which translated into enhanced enrichment of non-rRNA transcripts and increased depth of RNA-seq coverage. The results indicated that, in addition to improving RNA-seq sensitivity, efficient rRNA removal enhanced detection of low abundance transcripts via qPCR. Finally, we demonstrate that the Ribo-Zero kit also exhibited the highest efficiency when P. aeruginosa/Staphylococcus aureus co-culture RNA samples were tested.
authors.affiliation: Department of Biological Sciences, Binghamton Biofilm Research Center, Binghamton University, Binghamton, NY, USA.
authors.forename: Olga E
authors.initials: OE
authors.lastname: Petrova
authors.affiliation: Roche Pharma Research and Early Development, Immunology, Inflammation and Infectious Diseases, Roche Innovation Center Basel, F. Hoffmann-La Roche Ltd, Grenzacherstrasse 124, 4070 Basel, Switzerland.
authors.forename: Fernando
authors.initials: F
authors.lastname: Garcia-Alcalde
authors.affiliation: Roche Pharma Research and Early Development, Immunology, Inflammation and Infectious Diseases, Roche Innovation Center Basel, F. Hoffmann-La Roche Ltd, Grenzacherstrasse 124, 4070 Basel, Switzerland.
authors.forename: Claudia
authors.initials: C
authors.lastname: Zampaloni
authors.affiliation: Department of Biological Sciences, Binghamton Biofilm Research Center, Binghamton University, Binghamton, NY, USA.
authors.forename: Karin
authors.initials: K
authors.lastname: Sauer
mesh_terms: D001419:Bacteria / Q000235:genetics*; D018407:Bacterial Physiological Phenomena; D018441:Biofilms; D020869:Gene Expression Profiling / Q000379:methods*; D015964:Gene Expression Regulation, Bacterial; D011550:Pseudomonas aeruginosa / Q000235:genetics / Q000502:physiology; D012329:RNA, Bacterial; D012680:Sensitivity and Specificity; D017423:Sequence Analysis, RNA / Q000379:methods*; D013211:Staphylococcus aureus
chemical_list: D012329:RNA, Bacterial