Discrimination of other non-coding RNAs from miRNAs by read alignment patterns. (a) VAMP visualization of read alignments to three murine miRNA hairpins, which have been recently experimentally validated as non-coding RNAs. In contrast to canonical miRNAs, reads do not align in non-overlapping blocks with uniform 5′ termini, as expected for mature miRNA reads. (b) ClustalW multiple read alignment of sequencing reads to mmu-mir-2133-2.

Comparison of total and unique read annotation as observed for Pool small RNAs. Pie charts were used for illustrating the distribution of read annotation as miRNA, Rfam ncRNA, piwiRNA (piRNA), repetitive rodent RNA, or unknown. Pie charts are given for the total read set (a) and the unique read set (b).

Quantitative analysis of miRNA transcription. The histogram plot in (a) shows that the majority of miRNAs exhibit read counts between 10 and 1.000. (b) Read counts were then normalized to the individual size of each library (in million read counts) and log₁₀-transformed to achieved a Gaussian distribution of miRNA expression across all 6 CHO cell lines, visualized using a boxplot diagram.

Identification and annotation of conserved CHO miRNAs. (a) Small RNA reads were mapped to the entire set of known miRNA hairpin sequences, in the form of a concatenated sequence leaving spacers of 50 bases (N₅₀) between each hairpin sequence (1). In the second step, miRNA isoforms (isomiRs) were grouped and further represented by the most abundant isomiR sequence (2). For annotation of miRNA reads, three scenarios were differentiated: mapping of both arms of the hairpin duplex (A); mapping of only one arm of the hairpin duplex (B) and mapping of regions adjacent to the duplex (C). For the visualization of short read alignments to the miRNA hairpin reference sequence, VAMP, a software developed at the Center for Biotechnology in Bielefeld, Germany was used: orange bars in the upper section represent annotated hairpin sequences while the lower section shows the single-basepair coverage computed from read alignments; green color indicates perfect coverage with no mismatches, yellow color best-match coverage (containing 1–3 mismatches), and red color represents the complete coverage (reads with 1–3 mismatches that were found to align to a different hairpin at lower mismatch rate). (b) The coverage pattern for hsa-miR-18b at single-basepair level is shown in: both hairpin arms are mapped at high perfect coverage, with more reads mapping to the 5′ arm of the hairpin. (c) A locus in the hairpin genome containing 9 miRNA hairpin sequences from Rattus norvegicus is shown at lower zoom: high perfect coverage is generally observed at the 5′ and 3′ duplex positions within a hairpin. In most cases a predominant hairpin-arm exists (high coverage), while in some cases (mir-106b) both hairpins-arms show equal coverage. In a few cases, antisense alignments (mir-96, mir-98) are observed, indicated by coverage facing downwards. (For interpretation of the references to color in text, the reader is referred to the web version of the article.)

Hairpin classification and Chinese hamster ovary miRNA conservation. (a) Bar chart showing total read counts over read length for the complete read set (dark) compared to reads that had mapped the comprehensive miRNA genome and can therefore be considered as conserved miRNA reads (bright). (b) Of 235 canoncial miRNA hairpins that were discovered in CHO cells, 105 miRNA had been mapped at either the 5′ (54) or 3′ (51) position, while 130 hairpins had been mapped at both hairpin arms. The ratio of 5′ and 3′ read abundances was calculated for these 130 hairpins, resulting in 44 instances where the 5p/3p ratio exceeded an arbitrary ratio cut-off of 20:1, while in 24 instances it was below 1:20. (c) Out of 224 miRNAs that showed perfect identity to miRBase miRNA sequences, 82% had a human, mouse, or rat ortholog. Among the remaining 18% that did not have a perfect human, or a rodent ortholog, cow, platypus, and chicken were the most frequently found species.

Prediction of novel miRNAs. Several criteria were defined for the identification of novel miRNA genes and are exemplarily shown for novel miRNA candidate IV: (a) previously reported descriptors were used in blockbuster (Langenberger et al., 2009b; van der Burgt et al., 2009) to identify genomic loci with miRNA-like alignment patterns such as “sharp” blocks with uniform 5′ termini and coverage of both hairpin-arms. (b) RNAfold was used for prediction of RNA secondary structures of these genomic regions. Sequences that did not fold in silico into miRNA hairpin-like structures were filtered and discarded. The remaining sequences between 40 and 170 nucleotides in length were sorted according to their genomic location (c). Short read sequences located in intergenic regions were subjected to a support vector machine that was trained to identify Dicer cleaved duplexes at a 90% recall rate. These were manually screened to identify 11 putative novel miRNAs, which are listed in table-format (d) giving the mouse genomic location of the cluster as well as locations of the most abundant 5′ and 3′ reads.

miRNA transcription provides information on the cellular state of CHO cell lines. (a) Cartoon depicting the biological relationship of sequenced CHO cell lines. (b) Unsupervised hierarchical clustering of CHO cell lines according to their miRNA transcription profiles identified 3 nodes, corresponding to serum-dependent K1 and DXB11 cell lines (1), serum-free adapted host and recombinant K1 cell lines (2), and serum-free host and recombinant DUXB11 cell lines (3). Principal component analysis of a miRNA expression matrix consisting of 6 samples (CHO cell lines) and 365 variables (conserved miRNAs) was centered and used for singular value decomposition using R. Principal components were retrieved, and biplot graphs were chosen for their illustration as PC1 versus PC2 (c) and PC2 versus PC3 (d).

Analysis of differential miRNA transcription in CHO cell lines. (a) Differential expression analysis for the contrast serum-free versus serum-dependent (one-way ANOVA, p ≤ 0.05) was performed considering only miRNAs with read counts > 500. Log₂ fold changes of 18 significantly regulated miRNAs are depicted in a bubble plot, where miRNAs are sorted according to mean expression levels, represented by the bubble size. (b) The significant reduction of miR-221-3p in serum-free adapted cells was accompanied by an overall switch of the ratio of 5′ and 3′ mature miRNA levels originating from mir-221 from positive to negative, wich was restored again in recombinant cell lines. (c) Differential expression analysis of miRNAs between recombinant and serum-free CHO host-cells (one-way ANOVA, p < 0.05, read count > 500) identified 8 significantly regulated miRNAs. (d) Six out of 18 miRNAs that were found regulated between serum-free and serum-dependent growth, and 4 miRNAs that were found regulated in recombinant versus host cells were chosen for qPCR validation. Log₂ transformed fold changes for both contrasts are given as bar chart, where black bars represent log₂ fold changes as determined by sequencing and grey bars as determined by quantitative PCR.