<p>log<sub>2</sub> distance in base pairs from associated canonical 3’SSs to (A) 1,117 significantly differentially used novel 3’SSs, (B) 16,673 novel 3’SSs with canonical intron motifs (GT/AG) used more highly in the mutants but not significant, and (C) 18,660 novel 3’SSs with canonical intron motifs (GT/AG) used more highly in the wild-types but not significant. Zero represents the position of the canonical 3’SS. Negative and positive distances indicate that the cryptic 3’SS is respectively upstream or downstream from the canonical 3’SS. Inset in (A) shows base-by-base binning from zero to 50 base pairs upstream of canonical 3’SS. Red and blue histograms represent junctions with significantly higher usage in <i>SF3B1</i> mutants or <i>SF3B1</i> wild-type samples, respectively. (D) Upper red and blue heatmap shows for each sample the log<sub>2</sub> library-normalized count <i>z</i>-score for 619 cryptic 3’SSs used significantly more often in the <i>SF3B1</i> mutants and located 10–30 bp upstream of canonical 3’SSs (DEXSeq, BH-adjusted <i>p</i> < 0.1). Grey bars at left indicate frequency of <i>SF3B1</i> mutant allele in RNA-seq data. Colorbars indicate <i>SF3B1</i> mutation status, cancer type, and whether the <i>SF3B1</i> mutation is located in the HEAT 5–9 repeats. Black and white colorbar indicates whether novel 3’SSs are out-of-frame (black) relative to canonical 3’SSs. Bottom green heatmap shows relative expression levels for the genes containing each cryptic 3’SS. We calculated the average expression of each gene in each cancer type and normalized by the maximum expression for each gene so that the maximum value in each column is one (see <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004105#sec010" target="_blank">Methods</a>). Cryptic 3’SSs not observed in all cancer types tend to have differing gene expression levels between cancers. (E) Locations and frequency of <i>SF3B1</i> mutations in HEAT repeats 5–9. Mutations observed more than once in COSMIC (upper axis) cluster in ~10 amino acid hotspots in each HEAT repeat; most frequent mutation in each hotspot is labeled. Bottom axis shows locations and frequency of mutations in our study. BRCA samples with A663V and Y765C mutations do not show evidence for cryptic 3’SS selection.</p
