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Direct cloning and parallel sequencing, an extremely powerful method for microRNA (miRNA) discovery, has not yet been applied to bacterial tran- scriptomes. Here we present sRNA-Seq, an unbi- ased 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 bacte- rial transcriptome. From 407039 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, character- ization 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 bac- teria has been greatly underestimated and that future efforts to analyze bacterial transcriptomes will benefit from direct cloning and parallel sequen- cing experiments aided by 5S/tRNA depletion.

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