A three-dimensional PCA plot of the N. furzeri MZM small RNA-Seq libraries of all three tissues (brain – red, liver – green, blue – skin) and all investigated ages (from light to dark: 5, 12, 20, 27, 39 weeks). Whereas the samples cluster well according to their tissue belongings, a distinct separation regarding the ages can only be observed for the youngest samples in each tissue. A PCA plot of the GRZ strain, can be found in Supplement Table 2

Annotation, expression profiles and prediction comparison for miR-499.We annotated the pre-miR-499 on sgr09, position 55,926,017– 55,926,134 and the two mature miRNAs at 55,926,048–55,926,069 and 55,926,085–55,926,106. The six methods used for miRNA detection are displayed, CID-miRNA was not able to detect this miRNA. Tools working independent of the small RNA-Seq data BLAST (cyan), Infernal (olive green) and goRAP (orange) vary in their annotation length. The latter two programs are based on covariance models, identifying mostly the complete pre-miRNA. The remaining two programs miRDeep* and Blockbuster are based on small RNA-Seq data (*) and therefore accurately annotate the mature miRNAs. MiR-499 is expressed weakly within N. furzeri MZM 12 month liver library and therefore could not be detected by miRDeep* and Blockbuster. In the N. furzeri MZM 12 month brain library, miR-499 was expressed strongs enough to be detected by both programs

Venn diagram of predicted miRNA genes from four tools miRDeep*, Infernal, goRap and BLAST. Only 2 of the 33 candidates predicted by CID-miRNA overlapped with any of the other miRNA candidates. Nevertheless, all 33 candidates were selected as miRNAs after manual inspectations. The total number of miRNA predictions after and before applying any filtering step are shown in brackets for each tool