Maximum TPR values were obtained at the following threshold: psRNATarget: 5 (Maximum expectation), Tapirfasta: 10 (Score), Tapirhybrid: 10 (Score), psRobot: 5 (Penalty score threshold), Target_Prediction: 4 (Penalty score), Targetfinder: 6.5 (Prediction Score), Target-align: 110 (Maximum Score), p-TAREF: 4 (SVR score), RNAhybrid: 1 (p-value).
psRNATarget program was executed with parameters at their default settings i.e. score or maximum expectation at 3, length of complementary scoring at 20, target accessibility at 25, flanking length around target 17 bp upstream and 13 bp downstream and keeping range of central mismatch leading to translational inhibition in between 9 11.
A minimal ProteinProspector protein score of 15, a peptide score of 15, a maximum expectation value of 0.1, and a minimal discriminant score threshold of 0.0 were used for initial identification criteria.
The parameters were as follows - Maximum expectation: 3, Length for complementarity scoring: 20, Allowed maximum energy to unpair the target site (UPE): 25, Flanking length around target site for target accessibility analysis: 17 bp, Range of central mismatch leading to translation inhibition: 9 11 nt.
Maximum expectation value for accepting individual peptide ion scores [−10*Log(p)] was set to ≤0.05, where p is the probability that the observed match is a random event.
We ran the known miRNAs, new miRNAs and 19 23 nt smRNAs against the TSS with a maximum expectation of 5 and an hspsize (which is the length of the region used to score the complementarity between the miRNA and its target) equal to the length of the smRNA (to ensure that the entire sequence of the smRNA is considered by the scoring algorithm).
Acceptance criteria for the reported results were as follows: minimal scores for proteins and peptides were 22 and 15, respectively, and maximum expectation values for proteins and peptides were 0.05 and 0.1, respectively.
The maximum expectation value (measures the complementarity between small RNA sequences and its target transcripts), the hsp size (length for complementary scoring) and target accessibility were set at 3.5, 17 and 25, respectively.
A maximum expectation value of 0.1 was employed as a threshold for peptide identifications.
We conducted the analyses under two settings: a strict setting where the maximum expectation was set to ≤2.0 (i.e., low probability of false positives) and a less stringent setting where sequences with a score >2.0 3.0 were allowed.
