Table 1 Aggregated Shortstack outputs for all sRNA libraries and the counts of sRNA clusters for each step

Not analyzed due to run in –nohp mode

No reads at all aligned in locus

DicerCall was invalid (<80% of reads in the Dicer size range defined by –dicermin and –dicermax)

Major RNA abundance was less than 2 reads

Major RNA length is not in the Dicer size range defined by –dicermin and –dicermax

Locus size is > than maximum allowed for RNA folding per option –foldsize (default is 300 nts)

Locus is not stranded (>20% and <80% of reads aligned to top strand)

RNA folding attempt failed at locus

Strand of possible mature miRNA is opposite to that of the locus

Retrieval of possible mature miRNA position failed

General failure to compute miRNA-star position

Possible mature miRNA had >5 unpaired bases in predicted precursor secondary structure

Possible mature miRNA was not contained in a single predicted vhairpin

Possible miRNA/miRNA* duplex had >2 bulges and/or >3 bulged nts v

Imprecise processing: Reads for possible miRNA, miRNA-star, and their 3p variants added up to less than 50% of the total reads at the locus

Maybe. Passed all tests EXCEPT that the miRNA-star was not sequenced. INSUFFICIENT evidence to support a de novo annotation of a new miRNA family

Yes. Passed all tests INCLUDING sequencing of the exact miRNA-star. Can support a de novo annotation of a new miRNA family