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Additional file 2: Figure S1. TMB and expressed TMB relative to sequencing coverage and tumor purity. Figure S2. Gene-wise mutation frequency among microsatellite stable CRCs. Figure S3. Gene set enrichment analyses of expressed mutations. Figure S4. Validation of mutation expression frequencies in TCGA. Figure S5. Heatmap of expressed mutations per gene and tumor sample. Figure S6. Logistic PCA of the expressed mutation matrix. Figure S7. Genes with frequent non-expressed mutations. Figure S8. Genes with frequent non-expressed mutated alleles. Figure S9. Genes with both expression and silencing of the mutated allele among tumors. Figure S10. Illustration of estimates used to describe relative allelic expression of mutations. Figure S11. RNA and DNA level MAFs in primary MSS CRCs in the in-house series. Figure S12. RNA MAF versus DNA MAF in TCGA. Figure S13. Genes with high relative allelic expression of mutations. Figure S14. RNA MAF versus DNA MAF in in-house series according to mutation/target gene category. Figure S15. Relative allelic mutation expression in TCGA data according to allelic copy number balance. Figure S16. Allele-specific expression of TP53 and KRAS mutations among 241 primary colon cancers in the TCGA validation series. Figure S17. Allele-specific expression of APC mutations in the in-house series of primary CRCs. Figure S18. Allele-specific mutation expression and drug sensitivity in CRC cell lines. Figure S19. Minimal evolutionary changes in mutation expression during metastasis. Figure S20. Comparisons of DNA MAFs among multiple tumor samples from each of 4 patients. Figure S21. Comparisons of RNA MAFs among multiple tumor samples from each of 4 patients. Figure S22. Comparisons of ΔMAF RNA|DNA among multiple tumor samples from each of 4 patients. Figure S23. Comparisons of RNA read counts of mutated alleles among multiple tumor samples from each of 4 patients.

Expression differences between colon-like and undifferentiated cell lines. a PCA plots show the spontaneous split between the two subgroups in all three datasets (mRNA, miRNA and protein). b Volcano plots show differentially expressed genes in undifferentiated (blue) versus colon-like (yellow) cell lines on the mRNA, miRNA and protein levels. Horizontal dashed lines mark the highest p-value that produces an adjusted p-value of

Outlier analysis reveals high level amplification events associated with marked mRNA expression changes, pointing to potential driver genes in individual cell lines. a A total of 280 genes were nominated, figure shows 15 nominated genes overlapping with the COSMIC Cancer Gene Census. Vertical axis shows gene expression values (log2 scale) and horizontal axis shows copy number aberration values (PCF values, log2 scale). The analysis pinpointed CNA values that were substantially higher in one or a few cell lines compared to remaining cell lines and hence 14 of the nominated genes were high amplification events, while one gene (SS18) was nominated on basis of all samples having loss except V9P. b Ten outlier genes had available associated protein data and for most genes the increase in gene expression was consistent on the protein level. Vertical axis shows relative protein expression (median centered normalized log2 values), horizontal axis shows gene expression values (log2 scale). (PDF 1233Â kb)

DNA level aberrations. a SNVs and indel counts in 34 cell lines. MSI cell lines generally displayed numerous SNVs/indels, in contrast to MSS cell lines, although DLD-1/HCT15 were less typical with a lower indel burden compared to remaining MSIs. b The percentage of the genome with aberrant CNA reflects MSI status rather than CMS subtype. The figure includes 29 unique MSI/MSS cell lines. c CMS frequency of CNAs. Vertical axis indicates frequency, horizontal axes shows chromosomes 1–22, separated by vertical lines (whole lines separates chromosomes, dashed lines separates chromosome arms). The most common gains in CMS2 (5 or more out of 9 CMS2 MSI/MSS cell lines) were found on 3q, 8q, 13q, 17q, 20p and 20q, while regions of loss were frequent on 1p, 3p, 4q, 6p, 6q, 8p, 16p, 16q, 17p, 18 p, 18q, 20p and 22q. In CMS4 the most common gains (4 or more out of 7 CMS4 MSI/MSS cell lines) were found on 3q, 5p, 5q, 7p, 7q, 12p, 20p, 20q and 22q, while losses were frequent on 3p, 4p, 4q, 6q, 15q, 17p, 18q and 22q. The plots for CMS2 and CMS4 are placed together for easier visual comparison. A frequency plot for CMS3 was included, but the low sample number limits interpretations of frequent alterations in this group. d Differential frequencies of CNAs in undifferentiated versus colon-like cell lines. The vertical axis indicates the frequency difference between undifferentiated – colon-like cell lines (i.e. the frequency in undifferentiated cell lines minus the frequency of aberration in colon-like cell lines). The horizontal axis indicates chromosomes 1–22 (chromosomes separated by whole lines, chromosome arms separated by dashed lines). Yellow areas represent regions with higher frequencies of CNAs in colon-like cell lines, purple areas represent regions with higher frequencies of CNAs in undifferentiated cell lines. CMS: consensus molecular subtype, CNA: copy number aberration, MSI: microsatellite instable, MSS: microsatellite stable, SNV: single nucleotide variant. (PDF 830 kb)