Differential expression in HD-ST (x-axis) and HD-FC (y-axis) samples. Black spots show the miRNA loci and gray spots show all the sequences (reference miRNAs and isomiRS) mapping onto the miRNA loci. The darker and lighter lines mark the limits of expression fold changes in HD versus control samples above 1.2 and below –1.2, respectively.

Percentage of miRNAs with nucleotide substitution variants, according to the nucleotide position. A scheme of the mature miRNA appears in the upper part of the figure showing the main regions involved in 3′-UTR gene targeting: 5′-seed (nt 2–8) the cleavage (nt 10–12) and anchor (nt 13–16) sites. A gray square highlights the less variable positions along the miRNA. The color pattern in the bars indicates the proportion of a specific type of nucleotide present in the isomiR, at every position: A (red), U (blue), C (green) and G (orange). In this analysis, nucleotide substitution variants with more than 50 counts in each library are considered.

Histogram displaying the percentage of miRNAs with different types of IsomiRs (5′-trimming, 3′-trimming, 3′-additions and nucleotide substitutions), in C-FC (1), HD-FC (2), C-ST (3) and HD-ST (4) small RNA libraries. In this analysis, IsomiRs with frequencies >10 were considered. For every type of variability, the histogram shows the proportion of miRNAs presenting one (white), two (gray) or more than two (black) IsomiRs (A). An example of a miRNA (miR-124) displaying two 5′-trimming variants (T1 and T2) is shown below. Arrows in the precursor miRNA (Pre-miR-124) point the beginning of each isomiR. The histogram in (B) shows the abundance of the IsomiRs with respect to the corresponding reference miRNA, for every type of variability. The five gray color intensities from dark to light indicate the frequency of the isomiR with respect to the reference miRNA: <20%; 20–40%; 40–60%; 60–80% and >80%. The example below shows a 3′-trimming variant (T1) with a relative abundance with respect to the reference miRNA (miR-103) of around 40%. The arrow in Pre-miR-103 points the site where the 3′-trimming variant ends.

Percentage of miRNAs with 5′-trimming variants (A), 3′-trimming variants (B) and 3′-addition variants (C) in C-FC (1), HD-FC (2), C-ST (3) and HD-ST (4) small RNA samples, according to the type and position of the nucleotide involved. In this analysis, IsomiRs with frequencies >10 were considered. Pre-miRNA slicing occurring at different positions upstream or downstream of the reference miRNA extremes (5′- and 3′-) is indicated by −3 to –1 and +1 to +3, respectively (A, B). In the 3′-addition variants (C), 1, 2 and 3 indicate the number of nucleotides added to the 3′-terminus of the miRNA. In the upper part of each panel, a scheme of miRNA is shown. The part of the sequence in black corresponds to the mature reference miRNA and the part in gray highlights the precursor sequence (A, B) or the nucleotides added in the 3′-addition variants (C). The orange arrows and box highlight the nucleotides being examined in the histogram below. The size of the arrow is proportional to the percentage trimming variants at the signaled position. The color pattern of the bars indicates the nucleotides involved in the trimming variants at every position: A (red), U (blue), C (green) and G (orange). Significant bias toward a nucleotide involved in the trimming variant is shown with a white asterisk. Below each histogram an example of a variant is shown in a gray box. The attached gray arrow indicates the specific type of IsomiR, within the histogram, being analyzed.