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Nucleic Acids Research Advance Access originally published online on February 17, 2009
Nucleic Acids Research 2009 37(6):e46; doi:10.1093/nar/gkp080
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Nucleic Acids Research, 2009, Vol. 37, No. 6 e46
© 2009 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Experimental discovery of sRNAs in Vibrio cholerae by direct cloning, 5S/tRNA depletion and parallel sequencing

Jane M. Liu1, Jonathan Livny2, Michael S. Lawrence3, Marc D. Kimball1, Matthew K. Waldor2 and Andrew Camilli1,*

1HHMI and Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, MA 02111, 2HHMI and Channing Laboratory, Boston, MA 02115 and 3Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA

*To whom correspondence should be addressed. Tel: +1 617 636 2144; Fax: +1 617 636 2175; Email: andrew.camilli{at}tufts.edu

Received October 21, 2008. Revised January 22, 2009. Accepted January 28, 2009.

Direct cloning and parallel sequencing, an extremely powerful method for microRNA (miRNA) discovery, has not yet been applied to bacterial transcriptomes. Here we present sRNA-Seq, an unbiased method that allows for interrogation of the entire small, non-coding RNA (sRNA) repertoire in any prokaryotic or eukaryotic organism. This method includes a novel treatment that depletes total RNA fractions of highly abundant tRNAs and small subunit rRNA, thereby enriching the starting pool for sRNA transcripts with novel functionality. As a proof-of-principle, we applied sRNA-Seq to the human pathogen Vibrio cholerae. Our results provide information, at unprecedented depth, on the complexity of the sRNA component of a bacterial transcriptome. From 407 039 sequence reads, all 20 known V. cholerae sRNAs, 500 new, putative intergenic sRNAs and 127 putative antisense sRNAs were identified in a limited number of growth conditions examined. In addition, characterization of a subset of the newly identified transcripts led to the identification of a novel sRNA regulator of carbon metabolism. Collectively, these results strongly suggest that the number of sRNAs in bacteria has been greatly underestimated and that future efforts to analyze bacterial transcriptomes will benefit from direct cloning and parallel sequencing experiments aided by 5S/tRNA depletion.


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