Evidence for the biogenesis of more than one thousand novel human miRNAs. (a) Number of human miRNAs in the miRBase database over time. NGS, next-generation sequencing. (b) Advantage of dataset pooling. Each of seven sequencing experiments detects a single strand from a miRNA gene (in blue). When the datasets are analyzed separately (top), the single sequencing read does not constitute evidence of miRNA Dicer processing as opposed to random degradation. However, when the datasets are pooled (bottom), the numbers and positions of the reads constitute strong evidence of Dicer processing. (c) Synergistic miRNA prediction pipeline. The workflow is described in the Results section. The three plots below the flowchart indicate the length of the sequences retained after each step; notice the approximately 22-nucleotide peak characteristic of Dicer processing. (d-h) Four key factors in miRNA biogenesis were silenced with RNA interference. sRNA expression was profiled with high-throughput sequencing in silenced and mock-transfected cells, and the log2 fold-change shown. The curves indicate the cumulative fraction of RNAs with the indicated fold-change or lower. The numbers in the left margins indicate the fractions of miRNAs that are substantially down-regulated (>30% change). Control sequences comprise transfer RNAs (tRNAs), small nucleolar RNAs (snoRNAs) and miscellaneous RNAs (miscRNAs) (in grey). *P <0.01; **P <0.001. (i-k) RNAs interacting with DGCR8, Ago1 and Ago2 have previously been detected by crosslinking immunoprecipitation coupled with high-throughput sequencing (CLIP-seq) [22–24]. For each known or novel miRNA the overlaps with these RNAs were plotted as the difference between the 5′ end of the miRNA hairpin and the interacting RNA. The number of miRNAs that are supported by CLIP-seq evidence is shown above each subfigure. In the cases where more than one interacting RNA supported a given miRNA, one random overlap was chosen, such that each data point represents one miRNA. The blue bars indicate the consensus positions of the miRNA strands.