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

Olga E. Petrova,Fernando Garcia-Alcalde,Claudia Zampaloni,Karin Sauer

doi:10.1038/srep41114

Olga E. Petrova, Fernando Garcia-Alcalde + Show 2 more

Open Access

https://doi.org/10.1038/srep41114

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Journal: Scientific Reports	Publication Date: Jan 24, 2017
Citations: 58	License type: open-access

Affiliation: Binghamton University, Roche (Switzerland)

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.

Highlights

Studies of regulatory networks in bacteria often utilize analyses of gene expression
In order to ensure valid comparison of the Ribosomal RNA (rRNA) depletion methods, the procedures were performed on aliquots of the same input RNA sample, with the comparisons repeated using biological triplicates
While signal saturation can be achieved with a total of ~3 million reads following Ribo-Zero rRNA depletion, samples treated with the RiboMinus or MICROBExpress kits will require in excess of 5 million reads to reach similar sequencing depth (Fig. 4G). These findings indicated that the Ribo-Zero rRNA depletion results in both higher reproducibility and sequencing depth compared to the MICROBExpress and RiboMinus treatments

Summary

Introduction

Studies of regulatory networks in bacteria often utilize analyses of gene expression. Such kits have been used on single-species cultures, multi-species communities, and environmental samples The efficiency of these methods, has varied in a manner dependent on bacterial species and sample composition, with significant carryover of rRNA often observed following RNA-seq data analysis, with up to 50% of reads corresponding to rRNA11–13. Approaches to address such shortcomings have included repeated rounds of subtractive hybridization, combination of different methods, or the design and synthesis of custom capture oligonucleotides[11,12]. The Ribo-Zero kit exhibited superior rRNA depletion efficiency when P. aeruginosa planktonic and P. aeruginosa/Staphylococcus aureus co-cultures samples were tested

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