Fig. 4
LncRNAs are more variably expressed than mRNAs. a, b Genome wide inter-individual variability (normalized standard deviation between expression of each transcript/locus in granulocytes from seven donors) of de novo granulocyte lncRNA (green) and mRNA (blue) transcripts (a) and loci (b). Donor expression level is averaged from three replicates (***P <10–16). Median values: lncRNA transcripts: 0.29, mRNA transcripts: 0.15, lncRNA loci: 0.26, mRNA loci: 0.15. c LncRNA inter-individual expression variability allows correct clustering (normalized level among seven donors) of three time points per donor. Maximum transcript expression among all 21 samples is set to 1 (red), minimum is 0 (white). Clustering was performed using pheatmap function in R (clustering_distance_rows = ‘euclidean’, clustering_distance_cols = ‘correlation’). Only transcripts detected (RPKM >0.2) in at least one of the total RNA-seq samples were analyzed. Chromosomes X, Y were discarded. d Significance of granulocyte de novo lncRNA and mRNA expression variability in seven donors assessed by ANOVA test (the three time points are used as replicates). Bars show the percentage of significantly variable lncRNA (green) and mRNA (blue) transcripts (left) and loci (right). Criteria for calling a transcript/locus ‘significantly variable’: ANOVA test P value <0.01, FDR (Benjamini-Hochberg correction) <0.05, fold change between highest and lowest expression in seven donors >3. Only transcripts/loci with RPKM >0.2 in at least one donor are included. Chromosomes X and Y were discarded from the analysis. Total number analyzed: lncRNA transcripts 4,464, mRNA transcripts 119,412, lncRNA loci 658, mRNA loci 5,797. e Example of a significantly variable transcript from lncRNA locus gra896. Top: an alternative gra896 TSS overlaps the publicly-annotated lncRNA RP11-1008C21.1 locus. Underneath: normalized total RNA-seq signal for three replicates of four donors scaling from -0.001 (reverse strand, light gray) to 0.004 (forward strand, black). Calculated expression level of the annotated lncRNA transcript marked with * is shown for each RNA-seq track. Significance result for this transcript among seven donors: ANOVA test P = 10–7, FDR (Benjamini-Hochberg) = 10–6, expression fold change = 5.2). f Bidirectional lncRNA transcripts show reduced expression variability. Boxplots show inter-individual variability of lncRNA transcripts split according to their position relative to protein-coding genes as in Fig. 1c. Median normalized standard deviation values: bidirectional: 0.22, antisense: 0.29, intergenic: 0.30. Dashed blue line indicates median expression variability of all de novo mRNA transcripts. g Inter-individual expression variability is lower for known ‘in PA’ lncRNA transcripts compared to those newly annotated in granulocytes (‘not in PA’ and ‘isoform not in PA’). Median normalized standard deviation values: ‘not in PA’: 0.33, ‘isoform not in PA’: 0.30, ‘in PA’: 0.24. Dashed blue line indicates median expression variability of all de novo mRNA transcripts. Remarks to boxplots a, b, c, g: Transcripts/loci not expressed (RPKM <0.2) in any of seven donors (total RNA-seq data) and data from chromosomes X, Y were discarded and outliers are not displayed. The box plot displays the full population but P value is calculated using Mann–Whitney U test on equalized sample size. n.s. not significant, ***P <10–16