Supplementary Table S1. Pyrosequencing primers used to quantify the methylation status of selected CG sites. CpG Probe ID Primer sequence Target Region coordinate Fwd: AGGAGGAGGGAATGTAAAATAGA chr3:148,415,620- AGTR1 149898314 Rev: ACTACCTAAAATCCTAACTATCAAAC 148,415,697 Seq: AGGGAATGTAAAATAGAGT Fwd: AGGGAATTTTGGATTAGTAATTTGA chr6:94,129,308- EPHA7 94185987 Rev: CTTACTCCACACTCCAATAATATCA 94,129,484 Seq: GGAATTTTGGATTAGTAATTTGAT Fwd: TTGTTAGAGGAGGGTGGAGAGG chr4:20,255,253- SLIT2 19864444 Rev: ACAAAAACAACATCTACCAACCA 20,255,449 Seq: TTTGGTATTTTGGGTTG Fwd: AGGGGATGTTTTGTTTTTATTAGAG chr6:133,562,774- EYA4 133603412 Rev: TTCCCRAAAAATTTAAAAATTCTCTCAACT 133,562,943 Seq: GTTTTGTTTTTATTAGAGGTATAG Fwd:GGAATTTTTAGGGAAGTTTTAGGTTAAAGA chr7:116,963,543- WNT2 116750796 Rev: TACAAATACTAACTCTTCATCCCAACTTCA 116,963,716 Seq: GGAAGTTTTAGGTTAAAGAGTA Fwd: AGTTGAGTTAGGGGGTTTAGG chr17:75,369,482- SEPT9 Rev: AACAACCAACCCAACACC 75369562 75,369,637 Seq: GTTAGTTTTGTATTGTAGGAG Fwd: AGTTTTAGGTTTYGGAGTAGGAAGGTT chr10:17,271,103- Rev: AAAATCCCCTCCCACTACCAT 17271192 VIM 17,271,233 Seq: GTTTTTAGTTYGGTTTTTATTGG Supplementary Table S2. Selected candidates: probes selected as candidates for colorectal cancer diagnostic markers based on microarray data. Normal Tumor tissue tissue Probe Name Gene cg CpG t-test Chromosome Annotation Product Mean Mean Symbol number coordinate p-value (Standard (Standard deviation) deviation) Homo sapiens angiotensin angiotensin II II receptor, type 1 receptor, AGTR1_P41_F AGTR1 cg22498996 3 149898314 0.063 (0.003) 0.481 (0.033) 2.48E-21 (AGTR1), transcript type 1 variant 1, mRNA. Homo sapiens EPH ephrin receptor EPHA7_E6_F EPHA7 cg20253378 6 94185987 receptor A7 (EPHA7), EphA7 0.057 (0.002) 0.303 (0.028) 1.27E-13 mRNA. Homo sapiens wingless- wingless-type MMTV type MMTV integration integration site family WNT2_P217_F WNT2 cg02655774 7 116750796 0.070 (0.003) 0.495 (0.037) 1.40E-19 site family member 2 member 2 precursor (WNT2), mRNA. Homo sapiens eyes eyes absent 4 absent homolog 4 isoform a EYA4_E277_F EYA4 cg23916819 6 133604483 (Drosophila) (EYA4), 0.185 (0.011) 0.839 (0.02) 5.16E-41 transcript variant 1, mRNA. slit homolog 2 Homo sapiens slit homolog 2 SLIT2_E111_R SLIT2 cg26860620 4 19864444 0.113 (0.007) 0.653 (0.025) 2.61E-35 (Drosophila) (SLIT2), mRNA. Supplementary Table S3. Differential methylation results in microarray data: loci found to be highly hypermethylated in tumor (mean difference greater than 0.20). Standard Standard Mean Mean CpG deviation deviation t-test Probe Name CG number Chromosome Gene in Normal in Tumor Coordinate in Normal in Tumor p-value tissue tissue tissue tissue EYA4_E277_F cg23916819 6 133604483 EYA4 0.185 0.011 0.839 0.024 5.16E-41 SLIT2_E111_R cg26860620 4 19864444 SLIT2 0.113 0.007 0.653 0.025 2.61E-35 HS3ST2_E145_R cg22228897 16 22733506 HS3ST2 0.188 0.008 0.712 0.023 1.04E-34 NPY_E31_R cg25884711 7 24290365 NPY 0.130 0.008 0.664 0.025 1.06E-34 TMEFF2_P152_R cg10348923 2 192768041 TMEFF2 0.139 0.006 0.628 0.024 1.73E-34 NPY_P295_F cg20666532 7 24290039 NPY 0.321 0.016 0.843 0.020 1.17E-33 SLIT2_P208_F cg13875122 4 19864125 SLIT2 0.143 0.008 0.642 0.025 7.44E-33 SFRP1_P157_F cg15881237 8 41286294 SFRP1 0.129 0.009 0.704 0.030 8.24E-33 SFRP1_E398_R cg27086426 8 41285739 SFRP1 0.111 0.007 0.633 0.027 1.28E-32 TFPI2_P152_R cg12005526 7 93358153 TFPI2 0.126 0.007 0.653 0.026 1.97E-32 GABRB3_P92_F cg16084228 15 24570112 GABRB3 0.346 0.012 0.716 0.020 1.04E-30 TFPI2_P9_F cg16934178 7 93358010 TFPI2 0.106 0.009 0.629 0.028 1.27E-30 TWIST1_E117_R cg03005254 7 19123703 TWIST1 0.079 0.003 0.680 0.034 3.28E-30 NGFB_P13_F cg11660906 1 115682393 NGFB 0.135 0.006 0.626 0.028 6.55E-30 TWIST1_P355_R cg01202666 7 19124175 TWIST1 0.176 0.007 0.576 0.023 2.02E-28 EYA4_P794_F cg24842760 6 133603412 EYA4 0.163 0.007 0.636 0.028 6.33E-28 DBC1_E204_F cg06602847 9 121171318 DBC1 0.211 0.008 0.639 0.024 1.23E-27 TWIST1_P44_R cg12926104 7 19123864 TWIST1 0.133 0.006 0.631 0.030 8.17E-27 RASGRF1_E16_F cg16752670 15 77169879 RASGRF1 0.077 0.004 0.513 0.028 1.35E-26 TMEFF2_E94_R cg10026428 2 192767795 TMEFF2 0.051 0.002 0.574 0.034 2.01E-26 THY1_P149_R cg18809507 11 118799239 THY1 0.175 0.009 0.572 0.025 2.21E-26 NTSR1_P318_F cg03567830 20 60810316 NTSR1 0.179 0.008 0.668 0.031 3.83E-26 FLT4_P180_R cg20020551 5 180009352 FLT4 0.057 0.002 0.559 0.033 5.30E-26 SOX17_P287_R cg19346665 8 55532761 SOX17 0.317 0.016 0.704 0.023 7.89E-26 GSTM2_E153_F cg07246306 1 110012367 GSTM2 0.236 0.018 0.716 0.028 1.36E-25 FLI1_E29_F cg20448922 11 128069228 FLI1 0.152 0.012 0.623 0.031 1.51E-25 HS3ST2_P171_F cg03823653 16 22733190 HS3ST2 0.107 0.007 0.517 0.029 3.67E-25 KDR_P445_R cg04695981 4 55686964 KDR 0.205 0.010 0.577 0.024 3.75E-25 NPY_P91_F cg20961293 7 24290243 NPY 0.042 0.001 0.377 0.023 4.50E-25 TPEF_seq_44_S88_R cg09698887 2 192767395 TMEFF2 0.173 0.008 0.581 0.027 5.58E-25 IGFBP3_P423_R cg12826145 7 45927819 IGFBP3 0.128 0.007 0.551 0.029 6.96E-25 SOX17_P303_F cg09626193 8 55532745 SOX17 0.268 0.015 0.604 0.026 1.77E-24 ALK_E183_R cg02931220 2 29997753 ALK 0.084 0.003 0.457 0.027 2.07E-24 ESR1_P151_R cg05171584 6 152170228 ESR1 0.436 0.017 0.801 0.019 2.23E-24 DLK1_E227_R cg20175079 14 100263209 DLK1 0.309 0.015 0.722 0.024 2.29E-24 NTRK3_P636_R cg25116345 15 86601301 NTRK3 0.147 0.008 0.515 0.025 2.52E-24 NTRK3_P752_F cg09147530 15 86601417 NTRK3 0.093 0.004 0.491 0.028 4.23E-24 MYOD1_E156_F cg20325846 11 17697891 MYOD1 0.249 0.012 0.608 0.022 7.61E-24 TFPI2_E141_F cg23686014 7 93357860 TFPI2 0.086 0.004 0.381 0.021 1.52E-23 ADCYAP1_P398_F cg01521573 18 894989 ADCYAP1 0.249 0.012 0.635 0.024 2.49E-23 ESR1_E298_R cg17191385 6 152170677 ESR1 0.114 0.006 0.501 0.028 3.00E-23 HTR1B_E232_R cg26034501 6 78229607 HTR1B 0.313 0.013 0.685 0.023 3.73E-23 GAS7_E148_F cg20649212 17 10042445 GAS7 0.053 0.003 0.505 0.033 4.30E-23 NGFB_E353_F cg02459758 1 115682027 NGFB 0.069 0.003 0.542 0.035 6.19E-23 CD40_P372_R cg26940394 20 44179941 CD40 0.114 0.007 0.547 0.033 7.36E-23 ADAMTS12_E52_R cg13887940 5 33927829 ADAMTS12 0.168 0.007 0.504 0.024 7.65E-23 CDH13_P88_F cg08977371 16 81217991 CDH13 0.286 0.012 0.701 0.030 7.66E-23 FLT4_E206_F cg17414561 5 180008966 FLT4 0.076 0.004 0.303 0.018 8.45E-23 HOXA5_P1324_F cg05640017 7 27151136 HOXA5 0.092 0.006 0.493 0.030 1.85E-22 SCGB3A1_E55_R cg19421379 5 179951038 SCGB3A1 0.153 0.009 0.576 0.031 2.30E-22 TGFB2_E226_R cg20490551 1 216586717 TGFB2 0.319 0.015 0.745 0.029 1.49E-21 AGTR1_P41_F cg22498996 3 149898314 AGTR1 0.063 0.003 0.481 0.033 2.48E-21 ER_seq_a1_S60_F cg16246567 6 152170860 ESR1 0.091 0.005 0.458 0.029 2.87E-21 HOXA5_E187_F cg12041264 7 27149625 HOXA5 0.539 0.023 0.881 0.020 3.25E-21 KDR_E79_F cg10740902 4 55686440 KDR 0.159 0.011 0.478 0.026 4.03E-21 DAB2IP_E18_R cg13907787 9 123501488 DAB2IP 0.053 0.006 0.487 0.034 4.25E-21 GSTM2_P453_R cg11063364 1 110011761 GSTM2 0.426 0.017 0.730 0.018 4.98E-21 CSPG2_E38_F cg21708808 5 82803377 CSPG2 0.172 0.008 0.577 0.030 5.54E-21 MYOD1_P50_F cg27060180 11 17697685 MYOD1 0.255 0.011 0.601 0.025 5.99E-21 FGF5_P238_R cg23520103 4 81406528 FGF5 0.080 0.005 0.515 0.036 1.08E-20 TBX1_P885_R cg03402455 22 18123341 TBX1 0.124 0.005 0.517 0.030 1.86E-20 PENK_P447_R cg07433146 8 57521590 PENK 0.391 0.019 0.712 0.023 3.21E-20 ALK_P28_F cg14528334 2 29997964 ALK 0.054 0.002 0.417 0.030 5.45E-20 NTRK3_E131_F cg00865584 15 86600534 NTRK3 0.054 0.002 0.404 0.029 7.91E-20 MOS_E60_R cg09241196 8 57189035 MOS 0.494 0.020 0.831 0.019 1.04E-19 WNT2_P217_F cg02655774 7 116750796 WNT2 0.070 0.003 0.495 0.037 1.40E-19 NRG1_E74_F cg25833018 8 32525369 NRG1 0.063 0.002 0.323 0.022 1.70E-19 GABRB3_E42_F cg20421990 15 24569978 GABRB3 0.137 0.008 0.429 0.024 3.45E-19 WT1_E32_F cg20134916 11 32413631 WT1 0.104 0.005 0.532 0.037 3.70E-19 EYA4_P508_F cg19148393 6 133603698 EYA4 0.076 0.007 0.415 0.028 4.30E-19 FGF5_E16_F cg08153517 4 81406782 FGF5 0.045 0.001 0.415 0.032 5.13E-19 HS3ST2_P546_F cg23911889 16 22732815 HS3ST2 0.082 0.004 0.351 0.022 6.96E-19 FGF3_P171_R cg15119027 11 69343300 FGF3 0.165 0.007 0.528 0.031 1.61E-18 EPHA5_E158_R cg25798792 4 66217946 EPHA5 0.074 0.005 0.386 0.027 1.66E-18 IGF2AS_E4_F cg16130058 11 2118327 IGF2AS 0.048 0.002 0.316 0.024 1.94E-18 CDH13_E102_F cg15047333 16 81218181 CDH13 0.101 0.004 0.470 0.032 3.18E-18 TUSC3_P85_R cg17170317 8 15442016 TUSC3 0.185 0.012 0.496 0.025 3.60E-18 CCNA1_P216_F cg05780306 13 35904417 CCNA1 0.203 0.014 0.496 0.027 5.82E-18 NRG1_P558_R cg12863621 8 32524737 NRG1 0.070 0.003 0.387 0.029 6.03E-18 GSTM2_P109_R cg25855733 1 110012105 GSTM2 0.134 0.009 0.443 0.027 1.02E-17 BDNF_P259_R cg18882544 11 27700131 BDNF 0.362 0.012 0.673 0.026 1.48E-17 WT1_P853_F cg08219028 11 32414516 WT1 0.110 0.007 0.421 0.030 1.72E-17 CDH11_P354_R cg13126606 16 63713774 CDH11 0.207 0.008 0.543 0.030 3.07E-17 OPCML_E219_R cg19151121 11 132907394 OPCML 0.142 0.007 0.448 0.029 3.60E-17 ADCYAP1_P455_R cg01799816 18 894932 ADCYAP1 0.090 0.004 0.383 0.027 3.98E-17 FLT3_E326_R cg18969328 13 27572379 FLT3 0.131 0.008 0.539 0.038 5.03E-17 THY1_P20_R cg11980653 11 118799110 THY1 0.120 0.005 0.343 0.021 6.45E-17 NEFL_P209_R cg05876319 8 24870155 NEFL 0.432 0.016 0.739 0.025 7.43E-17 HIC-1_seq_48_S103_R cg20066299 17 1907679 HIC1 0.344 0.017 0.697 0.027 7.69E-17 DBC1_P351_R cg14445814 9 121171873 DBC1 0.152 0.008 0.451 0.028 1.32E-16 TUSC3_E29_R cg10249582 8 15442130 TUSC3 0.343 0.021 0.690 0.026 1.47E-16 MME_E29_F cg20228377 3 156280182 MME 0.096 0.006 0.521 0.042 1.50E-16 ALPL_P433_F cg23352747 1 21708023 ALPL 0.353 0.013 0.619 0.022 1.82E-16 CYP1B1_E83_R cg09991178 2 38156713 CYP1B1 0.093 0.004 0.461 0.036 3.39E-16 DCC_P471_R cg22055405