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Validation of two ribosomal RNA removal methods for microbial metatranscriptomics


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.

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Figure 1: Technical reproducibility.
Figure 2: Effectiveness of bulk rRNA removal.
Figure 3: Enrichment of mRNA in the synthetic communities.
Figure 4: Fidelity of mRNA relative abundance.


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We thank M. Allgaier, T. Zhan, M. Hess and E. DeLong for helpful discussions and feedback, H.-P. Klenk (German Collection of Microorganisms and Cell Cultures), T. Zhang and F. Warnecke (Joint Genome Institute) for providing microbial biomass, L. Pennacchio for supporting and facilitating this project, and members of research and development and production teams at the Joint Genome Institute for sequencing support. S.H. and P.H. were supported by a grant from the Energy Biosciences Institute. R.S. was supported, in part, by the Israel Science Foundation Focal Initiatives in Research in Science and Technology program (1615/09), the EMBO Young Investigator program, the Minerva Foundation and the Yeda-Sela Center for basic research. O.W. was supported by the Kahn Center for Systems Biology of the Human Cell and an Azrieli Foundation Fellowship. The work was performed in part under the auspices of the US Department of Energy's Office of Science, Biological and Environmental Research Program, and by the University of California, Lawrence Berkeley National Laboratory under contract DE-AC02-05CH11231, Lawrence Livermore National Laboratory under contract DE-AC52-07NA27344 and Los Alamos National Laboratory under contract DE-AC02-06NA25396.

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S.H., K.S., S.G.T., F.C., E.A.L. and P.H. planned the experiments, S.H., K.S. and S.Y. executed the experiments, S.H., O.W., J.L.F., Z.W., R.S. and P.H. performed the data analysis and S.H. and P.H. wrote the manuscript.

Corresponding author

Correspondence to Philip Hugenholtz.

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The authors declare no competing financial interests.

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Supplementary Figures 1–3, Supplementary Tables 1–6, Supplementary Notes 1–4 (PDF 685 kb)

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He, S., Wurtzel, O., Singh, K. et al. Validation of two ribosomal RNA removal methods for microbial metatranscriptomics. Nat Methods 7, 807–812 (2010).

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