The machine used for the pre-analysis was a HP Workstation xw9300 with a Dual Core AMD Opteron(tm) processor 275 2194.15 MHz and 8 Gb of RAM memory. We have used the stand-alone version for the adapter recognition step allowing only three mismatches and no gaps. The minimal size of the adapter recognized was set up to 10 nucleotides. The time process was 30 min for 6 millions reads. After that, sequences were annotated using human pre-miRNA and mature miRNA databases provided by the miRBase (http://microrna.sanger.ac.uk/sequences/ ) available at the SeqBuster server (15 min required). In addition, using the stand-alone version, the data were also mapped onto mRNA and genome databases (1 and 2.5 h required, respectively) (http://hgdownload.cse.ucsc.edu/goldenPath/hg18/bigZips/ ). The annotated files were uploaded to the server and stored for subsequent analyses.
For precursor-miRNAs annotation, the following parameters were configured: one mismatch, three nucleotides in the 3′ addition variants and the priority degree equal to 3. For miRNAs and miRNA*annotation, the following parameters were configured: one mismatch, three nucleotides in the 3′ or 5′ trimming variants, three nucleotides in the 3′ addition variants, a priority degree equal to 1 and 2 for the miRNA and the miRNA* databases, respectively, and the parental database was the precursor miRNA database. These options permitted the annotations of the following alignments: (i) perfect match, where the sequence is completely identical to the reference sequence; (ii) trimming at the 3′-end of the reference miRNA sequence, which is a miRNA variant several nucleotides shorter or longer that matches to the mature or precursor reference sequence, respectively; (iii) trimming at the 5′-end of the sequence, an analogous case focused in the 5′-end of the miRNA; (iv) nucleotide additions at the 3′-end of the sequence and (v) nucleotide substitutions, showing nucleotide modifications with respect to the reference sequence. The parameters for the alignment in mRNA and genome databases allowed as much as one mismatch and up to three nucleotide additions in the 3′-terminus. The priority parameter was equal to 4 and 5 for the mRNA and genome databases, respectively.
For precursor-miRNAs annotation, the following parameters were configured: one mismatch, three nucleotides in the 3′ addition variants and the priority degree equal to 3. For miRNAs and miRNA*annotation, the following parameters were configured: one mismatch, three nucleotides in the 3′ or 5′ trimming variants, three nucleotides in the 3′ addition variants, a priority degree equal to 1 and 2 for the miRNA and the miRNA* databases, respectively, and the parental database was the precursor miRNA database. These options permitted the annotations of the following alignments: (i) perfect match, where the sequence is completely identical to the reference sequence; (ii) trimming at the 3′-end of the reference miRNA sequence, which is a miRNA variant several nucleotides shorter or longer that matches to the mature or precursor reference sequence, respectively; (iii) trimming at the 5′-end of the sequence, an analogous case focused in the 5′-end of the miRNA; (iv) nucleotide additions at the 3′-end of the sequence and (v) nucleotide substitutions, showing nucleotide modifications with respect to the reference sequence. The parameters for the alignment in mRNA and genome databases allowed as much as one mismatch and up to three nucleotide additions in the 3′-terminus. The priority parameter was equal to 4 and 5 for the mRNA and genome databases, respectively.
