The predominance of rRNAs in the transcriptome is a major technical challenge in sequence-based analysis of cDNAs from microbial isolates and communities. Several approaches have been applied to deplete rRNAs from (meta)transcriptomes, but no systematic investigation of potential biases introduced by any of these approaches has been reported. Here we validated the effectiveness and fidelity of the two most commonly used approaches, subtractive hybridization and exonuclease digestion, as well as combinations of these treatments, on two synthetic five-microorganism metatranscriptomes using massively parallel sequencing. We found that the effectiveness of rRNA removal was a function of community composition and RNA integrity for these treatments. Subtractive hybridization alone introduced the least bias in relative transcript abundance, whereas exonuclease and in particular combined treatments greatly compromised mRNA abundance fidelity. Illumina sequencing itself also can compromise quantitative data analysis by introducing a G+C bias between runs.
At a glance
- Prokaryotic transcriptomics: a new view on regulation, physiology and pathogenicity. Nat. Rev. Genet. 11, 9–16 (2010). &
- Chemical composition of Escherichia coli . in Escherichia coli and Salmonella: Cellular and Molecular Biology 2nd edn., vol. 1 (eds. Neidhardt, F.C. et al.) 13–17 (ASM Press, Washington, D.C., 1996). &
- RNA: protein ratio of the unicellular organism as a characteristic of phosphorous and nitrogen stoichiometry and of the cellular requirement of ribosomes for protein synthesis. BMC Biol. 4, 30 (2006). , , &
- Formation of mRNA 3′ ends in eukaryotes: mechanism, regulation, and interrelationships with other steps in mRNA synthesis. Microbiol. Mol. Biol. Rev. 63, 405–445 (1999). , &
- Simultaneous assessment of soil microbial community structure and function through analysis of the meta-transcriptome. PLoS ONE 3, e2527 (2008). et al.
- Identification of new genes in Sinorhizobium meliloti using the Genome Sequencer FLX system. BMC Microbiol. 8, 72 (2008). , , &
- A simple method to enrich mRNA from total prokaryotic RNA. Mol. Biotechnol. 10, 83–85 (1998). &
- Bacterial mRNA purification by magnetic capture-hybridization method. Microbiol. Immunol. 48, 91–96 (2004). et al.
- Microbial community gene expression in ocean surface waters. Proc. Natl. Acad. Sci. USA 105, 3805–3810 (2008). et al.
- Metatranscriptomics reveals unique microbial small RNAs in the ocean′s water column. Nature 459, 266–269 (2009). , &
- Transcription profiling-based identification of Staphylococcus aureus genes regulated by the agr and/or sarA loci. J. Bacteriol. 183, 7341–7353 (2001). et al.
- Isolation and analysis of mRNA from environmental microbial communities. J. Microbiol. Methods 75, 172–176 (2008). et al.
- Detection of large numbers of novel sequences in the metatranscriptomes of complex marine microbial communities. PLoS ONE 3, e3042 (2008). et al.
- Potential for phosphonoacetate utilization by marine bacteria in temperate coastal waters. Environ. Microbiol. 11, 111–125 (2009). et al.
- Metatranscriptomic array analysis of ′Candidatus Accumulibacter phosphatis′-enriched enhanced biological phosphorus removal sludge. Environ. Microbiol. 12, 1205–1217 (2010). et al.
- An antibody-based microarray assay for small RNA detection. Nucleic Acids Res. 34, e52 (2006). , , , &
- Transcriptional activity of paddy soil bacterial communities. Environ. Microbiol. 11, 960–970 (2009). , , &
- Mapping the Burkholderia cenocepacia niche response via high-throughput sequencing. Proc. Natl. Acad. Sci. USA 106, 3976–3981 (2009). et al.
- Transcriptional response of Silicibacter pomeroyi DSS-3 to dimethylsulfoniopropionate (DMSP). Environ. Microbiol. 9, 2742–2755 (2007). et al.
- Inter-specific interactions between carbon-limited soil bacteria affect behavior and gene expression. Microb. Ecol. 58, 36–46 (2009). &
- Microbial community gene expression within colonies of the diazotroph, Trichodesmium, from the Southwest Pacific Ocean. ISME J. 3, 1286–1300 (2009). et al.
- Comparative day/night metatranscriptomic analysis of microbial communities in the North Pacific subtropical gyre. Environ. Microbiol. 11, 1358–1375 (2009). et al.
- In situ transcriptomic analysis of the globally important keystone N2-fixing taxon Crocosphaera watsonii . ISME J. 3, 618–631 (2009). et al.
- A single-base resolution map of an archaeal transcriptome. Genome Res. 20, 133–141 (2010). et al.
- The RIN: an RNA integrity number for assigning integrity values to RNA measurements. BMC Mol. Biol. 7, 3 (2006). et al.
- Development and quantitative analyses of a universal rRNA-subtraction protocol for microbial metatranscriptomics. ISME J. 4, 896–907 (2010). , &
- Characterizing the mouse ES cell transcriptome with Illumina sequencing. Genomics 92, 187–194 (2008). , , &
- A large genome center's improvements to the Illumina sequencing system. Nat. Methods 5, 1005–1010 (2008). et al.
- Supplementary Text and Figures (688K)
Supplementary Figures 1–3, Supplementary Tables 1–6, Supplementary Notes 1–4