Abstract
BackgroundNext generation sequencing (NGS) technologies have revolutionized gene expression studies and functional genomics analysis. However, further improvement of RNA sequencing protocols is still desirable, in order to reduce NGS costs and to increase its accuracy. In bacteria, a major problem in RNA sequencing is the abundance of ribosomal RNA (rRNA), which accounts for 95-98% of total RNA and can therefore hinder sufficient coverage of mRNA, the main focus of transcriptomic studies. Thus, efficient removal of rRNA is necessary to achieve optimal coverage, good detection sensitivity and reliable results. An additional challenge is presented by microorganisms with GC-rich genomes, in which rRNA removal is less efficient.ResultsIn this work, we tested two commercial kits for rRNA removal, either alone or in combination, on Burkholderia thailandensis. This bacterium, chosen as representative of the important Burkholderia genus, which includes both pathogenic and environmental bacteria, has a rather large (6.72 Mb) and GC-rich (67.7%) genome. Each enriched mRNA sample was sequenced through paired-end Illumina GAIIx run in duplicate, yielding between 10 and 40 million reads. We show that combined treatment with both kits allows an mRNA enrichment of more than 238-fold, enabling the sequencing of almost all (more than 90%) B. thailandensis transcripts from less than 10 million reads, without introducing any bias in mRNA relative abundance, thus preserving differential expression profile.ConclusionsThe mRNA enrichment protocol presented in this work leads to an increase in detection sensitivity up to 770% compared to total RNA; such increased sensitivity allows for a corresponding reduction in the number of sequencing reads necessary for the complete analysis of whole transcriptome expression profiling. Thus we can conclude that the MICROBExpress/Ovation combined rRNA removal method could be suitable for RNA sequencing of whole transcriptomes of microorganisms with high GC content and complex genomes enabling at the same time an important scaling down of sequencing costs.
Highlights
Generation sequencing (NGS) technologies have revolutionized gene expression studies and functional genomics analysis
Our results show that the combination of the two kits leads to optimal results in terms of ribosomal RNA (rRNA) removal, without introducing a significant bias on relative mRNA abundances, and allow the sequencing of a GC rich bacteria transcriptome with less than 10 millions reads
Total RNA from B. thailandensis E264 was subjected to ribosomal RNA removal treatment using either the MICROBExpress Bacterial mRNA Enrichment Kit (Mex) or the Ovation Prokaryotic RNA-seq System (Ov) separately, or a combination of the two kits (Mex-Ov)
Summary
Generation sequencing (NGS) technologies have revolutionized gene expression studies and functional genomics analysis. A major challenge in RNA-seq applications in bacterial cells is the enrichment for all transcript species other than rRNA and tRNA. Papers describing the use of high-throughput sequencing for transcriptomics in bacteria have used mRNA enrichment methods usually based on depletion of rRNA and other RNAs [2,3,4], utilizing two alternative approaches: (i) hybridization capture of rRNAs by antisense oligonucleotides followed by pull down through binding to magnetic beads, (ii) degradation of processed RNA such as mature rRNA and tRNA by a 5′–3′ exonuclease that digests RNA species with a 5′-monophosphate end. The rRNA capture approach is the only method suitable for precise quantitative analysis As it is based on 16S and 23S rRNA specific capture probes, depletion efficiency of these kits varies between bacterial species. The Ribo-Zero kit, based on rRNA capture, proved to be very efficient both on pure cultures and on faecal samples, while preserving mRNAs relative abundance
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