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MicroRNAs (miRNAs) are a class of small RNAs of approximately 22 nucleotides in length that regulate eukaryotic gene expression at the post-transcriptional level (Ambros 2004; Bartel 2004; Filipowicz et al. 2008). They are transcribed as long precursor RNA molecules (pri-miRNAs) and are successively processed by two key RNAses, namely Drosha and Dicer, into their mature forms of ~22 nucleotides (Kim 2005; Kim et al. 2009). These small RNAs regulate gene expression by binding to target sites in the 3’ untranslated region of mRNAs (3’UTR). Recognition of the 3’UTR by miRNAs is mediated through complementary hybridization at least between nucleotides 2-8, numbered from the 5’ end (seed sequences) of the small RNAs, and complementary sequences present in the 3’UTRs of mRNAs (Ambros 2004; Bartel 2004; Zamore and Haley 2005). Perfect or nearly perfect complementarities between miRNAs and their 3’UTRs induce mRNA cleavage by the RNAinduced silencing complex (RISC), whereas imperfect base pair matching may induce translational silencing through various molecular mechanisms, namely inhibition of translation initiation and activation of mRNA storage in P-bodies and/or stress granules (Pillai et al. 2007). This class of small RNAs is well conserved between eukaryotic organisms, suggesting that they appeared early in eukaryotic evolution and play fundamental roles in gene expression control. Each miRNA may repress hundreds of mRNAs and regulate a wide variety of biological processes, namely developmental timing (Feinbaum and Ambros 1999; Lau et al. 2001), cell differentiation (Tay et al. 2008), immune response (Ceppi et al. 2009) and infection (Chang et al. 2008). For this reason, their identification is essential to understand eukaryotic biology. Their small size, low abundance and high instability complicated early identification, but these obstacles have been overcome by next generation sequencing approaches, namely the Genome SequencerTM FLX from Roche, the Solexa/Illumina Genome Analyzer and the Applied Biosystems SOLiDTM Sequencer which are currently being routinely used for rapid miRNA identification and quantification in many eukaryotes (Burnside et al. 2008; Morin et al. 2008; Schulte et al. 2010). As in other vertebrates, miRNAs control gene expression in zebrafish, since defective miRNA processing arrest development (Wienholds et al. 2003). Also, a specific subset of miRNAs is required for brain morphogenesis in zebrafish embryos, but not for cell fate determination or axis formation (Giraldez et al. 2005). In other words, miRNAs play an

Computational Tools for Identification of microRNAs in Deep Sequencing Data Sets