Fig. 4: m6A profiling in MOLM13 cells.
From: RNA modifications detection by comparative Nanopore direct RNA sequencing
A Sharkfin plot showing the absolute value of the Nanocompore logistic regression log odd ratio (GMM logit method with context 2, x-axis) plotted against its p-value (-log10, y-axis, see Material and Methods). Each point represents a specific kmer of a transcript. Red points are DRACH kmers. B Metagene plot showing the distribution of significant m6A sites identified by Nanocompore (blue) and miCLIP (red). C Genome browser screenshot showing METTL3-dependent m6A sites in the ACTB transcript. The p-value track reports the Nanocompore GMM+Logistic regression method (see Material and Methods). D–F As in C but showing the three most significant β-actin sites at higher magnification. The sequence reported at the bottom corresponds to the RNA sequence in the 3’ to 5’ orientation, as the ACTB transcript is encoded on the minus strand. The m6A consensus GGACU sequences are highlighted in red. G Sylamer plot showing kmer enrichment in Nanocompore significant sites. The x-axis reports all Nanocompore sites with p-value<0.5 ranked from the most to the least significant. The y-axis reports the uncorrected Sylamer hypergeometric p-value of enrichment (one-sided test) of a certain motif in the first x Nanocompore sites vs the rest. The vertical dotted line delineates Nanocompore sites with p-value<0.01 (to the left of the line). The red line corresponds to the combined p-value (Fisher’s method) of all DRACH kmers. H m6A miCLIP coverage of clusters of significant Nanocompore sites (GMM logit (context 2) p-value<0.01). The y-axis shows the mean input-normalised miCLIP counts across sites. Shaded regions on the plot represent the mean±the standard deviation at each position in the profile (WT miCLIP n = 4, KO n = 2). Both the mean and bounds were smoothed using loess regression with a span of 0.6. The difference between WT and KO in the windows 0+/-20nt is statistically significant (p-value = 7.90 × 10−11, Mann-Whitney test). I Plot showing the fraction of Nanocompore significant peaks supported by a varying number of miCLIP reads (x-axis) in WT MOLM13 cells.
