Additional file 1: of Novel reference genes in colorectal cancer identify a distinct subset of high stage tumors and their associated histologically normal colonic tissues

Table S1. Clinical information of 79 CRC pairs. The MSI and MSS information were available for 8 tumors. Table S2. Tumor information of 50 CRC pairs from TCGA. Table S3. 21 classical reference genes and their annotated functions. Table S4. Expression profiles of 21 classical reference genes in 79 CRC cohort. Table S5. Expression profiles of 42 colorectal reference genes in 79 CRC cohort. Table S6. 23 reference genes with the annotated functions of cellular cargo transportation. Table S7. 13 reference genes with the annotated functions of structural proteins. Table S8. 9 reference genes with the annotated functions of enzymes. Table S9. Determination of possible function of 8 reference genes through correlation analysis. Table S10. Identification of 8 RGCOEX genes based on their correlation with 5 novel reference genes. Table S11. Genes coexpressed with 6 reference genes. Table S12. Expression profiles of 8 RGCOEX genes. Table S13. Expression profiles of classical 21 reference genes in 50 CRCs (TCGA). Table S14. Expression profiles of novel colorectal 42 reference genes in 50 CRC pairs (TCGA). Table S15. Oncogenes and tumor suppressors coexpressed with 3 reference genes (RAB1B, ACTR2 and CLTC). Figure S1. NGS analysis of 15 genes pertaining to ribosome biogenesis in 79 CRCs. Figure S1a. Upregulation of 15 genes pertaining to ribosome biogenesis in 79 CRCs. These 15ribosome biogenesis related genes were identified from 1223 upregulated genes (average T/N > 2 fold, FDR < 0.05 (ANOVA)) by DAVID Bioinformatics Resources 6.8 ( https://david.ncifcrf.gov/ ) [11]. The 15 ribosome biogenesis genes are: D-Tyrosyl-TRNA Deacylase 1 (DTD1), Dyskerin Pseudouridine Synthase 1 (DKC1), GTP Binding Protein 4 (GTPBP4), Ribosomal RNA Processing 1B (RRP1B), Block Of Proliferation 1 (BOP1), DDB1 and CUL4 Associated Factor 13 (DCAF13), Nucleolar Protein (NOP2), Ribosomal RNA processing protein 1 (RRP1), Nucleolar And Coiled-Body Phosphoprotein 1 (NOLC1), Nucleophosmin 1(NPM1), Biogenesis Of Ribosomes 1 (BRIX1), Nucleoplasmin 3 (NPM3), Ribonucleoprotein 58 (NOP58), Ribosomal RNA Processing 9 (RRP9) and Ribosome Biogenesis Regulator Homolog 1 (RRS1). Figure S1b. Differential expression of 15 genes pertaining to ribosome biogenesis among 79 CRC pairs in HCA. Figure S1c. Differential expression of 15 genes pertaining to ribosome biogenesis among 79 CRC pairs in PCA. Figure S2. NGS analysis of 21 classical reference genes in 79 CRC pairs. Figure S2a. Upregulation trend of 21 genes classical reference genes in 79 CRC pairs. Figure S2b. Upregulation of GAPDH in a subset of LST and HST. Figure S2c. Up and Down regulation of ACTB in a subset of LST and HST. Figure S2d. Up and Down regulation of B2M in a subset of LST and HST. Figure S3. Expression of 8 reference genes between HST and LST. Figure S3a.Selection of 8 reference gene CVs between 53 high stage HSTs and 26 LSTs (t-test: P = 0.0003). Figure S3b. Fourfold downregulation of 8 reference genes in 15 out 17 of HSTs detected by PCA. Figure S3c. Fourfold downregulation of 8 reference genes in 5 out 7 of H/LSNs detected by PCA. For stacked log2 ratio plots, FPKMs of 79 tumors and 79 normal samples were only normalized by the mean of FPKM of 26 LSNs for each gene since HSNs were more likely to have dysregulation. Figure S4. Downregulation of 8 RGCOEX genes in a subset of tumors and normal controls. Figure S4a. Four fold downregulation of 8 RGCOEX genes in 12 out of 16 H/LSTs detected by PCA. Figure S4b. Downregulation of 8 RGCOEX genes in 5 out 7 in H/LSNs detected by PCA. For stacked log2 ratio plots, FPKMs of 79 tumors and 79 normal samples were only normalized by the mean of FPKM of 26 LSNs for each gene since HSNs were more likely to have dysregulation. Figure S5. Downregulation of 8 reference genes, their 8 correlated genes and 10 tumor related genes in normal samples and tumors. Figure S5a. Downregulation of 8 reference genes in 79 normal samples. Figure S5b. Downregulation of 8 reference genes in 79 tumors. Figure S5c. Downregulation of 8 RGCOEX genes in a subset of normal samples. Figure S5d. Downregulation of 8 RGCOEX genes in a subset of tumors. Figure S5e. Downregulation of 8 reference genes in 5 out 7 CRC pairs. Figure S5 f. Downregulation of 8 RGCOEX genes in 5 out 7 CRC pairs. Figure S6. NGS analysis of tumor related genes in HSN. Figure S6a. Upregulation of COL6A1, COL6A2, COL1A2 and COL1A1 in N8. Figure S6b. Upregulation of CXCR1 and CXCR2 in N8. Figure S6c. Upregulation of MYC and CDK4 in N58. Figure S6d. Upregulation of MMP2 and MMP14 in N8. Figure S7. PCA Simulated normalization of 15 ribosome biogenesis genes by 7 reference genes in 79 CRCs. Figure S7a. Ribosome biogenesis without normalization. Figure S7b. Ribosome biogenesis normalized by the hypothetical reference gene (FPKM =100 for all 158 samples, (CV = 0%). Figure S7c. Ribosome biogenesis normalized by the C1orf43 (CV = 16%). Figure S7d. Ribosome biogenesis normalized by the RAB7A (CV = 17%). Figure S7e. Ribosome biogenesis normalized by the HEBP2 (CV = 21%). Figure S7 f. Ribosome biogenesis normalized by the ACTB (CV = 34%). Figure S7 g. Ribosome biogenesis normalized by the TFRC (CV = 52%). Figure S7 h. Ribosome biogenesis normalized by the HSP90AB1 (CV = 75%). Figure S8. Weak agreement of 3 ribosome biogenesis gene expression profiles between NGS and qPCR in 20 CRC pairs. Figure S8a1. Comparison of NGS and qPCR of BOP1. Figure S8a2. Comparison of NGS and qPCR of DKC1. Figure S8a3. Comparison of NGS and qPCR of RRP1B. Figure S8b1. Correlation of NGS and qPCR of BOP1. Figure S8b2. Correlation of NGS and qPCR of DKC1. Figure S8b3. Correlation of NGS and qPCR of RRP1B. Figure S9. NGS analysis of 6 cancer hallmark genes expression in 50 CRCs (TCGA). Figure S9a. Upregulation of CCND1, CDK4 and MYC in 50 CRCs. Figure S9b. Downregulation of AQP8, GPX3 and CD177 in 50 CRCs. Figure S10. Co-downregulation of 3 reference genes with 3 tumor suppressors in 3 out of 6 H/SNs detected by PCA. (PPTX 3420 kb)