Laboratory, Division of Medical Sciences, National Cancer Centre, Singapore

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1 Supplementary Information Exome sequencing of liver fluke-associated cholangiocarcinoma Choon Kiat Ong 1,2*, Chutima Subimerb 1,2,3*, Chawalit Pairojkul 3, Sopit Wongkham 3, Ioana Cutcutache 4, Willie Yu 1,2,5, John McPherson 4, George E. Allen 1,2, Cedric Chuan Young Ng 1,2, Bernice Huimin Wong 1,2, Swe Swe Myint 1,2, Vikneswari Rajasegaran 1,2, Hong Lee Heng 1,2, Anna Gan 1,2, Zhi Jiang Zang 6,7, Alice Yingting Wu 4, Jeanie Wu 7, Ming Hui Lee 7, DaChuan Huang 1,2, Pauline Ong 1,2, Waraporn Chan-on 1,2,3, Yun Cao 8, Chao-Nan Qian 8, Kiat Hon Lim 9, Aikseng Ooi 10, Karl Dykema 10, Kyle Furge 10, Veerapol Kukongviriyapan 3, Banchob Sripa 3, Chaisiri Wongkham 3, Puangrat Yongvanit 3, P. Andrew Futreal 11, Vajaraphongsa Bhudhisawasdi 3,#, Steve Rozen 4,#, Patrick Tan 12,7,13,#, Bin Tean Teh 1,2,# 1 National Cancer Centre Singapore-Van Andel Research Institute Translational Research Laboratory, Division of Medical Sciences, National Cancer Centre, Singapore 2 Laboratory of Cancer Therapeutics, Division of Cancer and Stem Cell Biology, Duke-NUS Graduate Medical School, Singapore 3 Liver fluke and Cholangiocarcinoma Research Center, Faculty of Medicine, Khon Kaen University, Khonkaen, Thailand 4 Division of Neuroscience and Behavioral Disorders, Duke-NUS Graduate Medical School, Singapore 5 National University of Singapore Graduate School for Integrative Sciences and Engineering, Singapore 6 Division of Cellular and Molecular Research, National Cancer Centre, Singapore 7 Division of Cancer and Stem Cell Biology, Duke-NUS Graduate Medical School, Singapore 8 Sun Yat-sen University Cancer Center, Guangzhou, P. R. China 9 Department of Pathology, Singapore General Hospital, Singapore 10 Laboratory of Computational Biology, Van Andel Research Institute, Grand Rapids, Michigan, USA 11 Cancer Genome Project, Wellcome Trust Sanger Institute, Hinxton, UK 12 Cancer Science Institute of Singapore, National University of Singapore, Centre for Life Sciences, Singapore 13 Genome Institute of Singapore, Singapore #Corresponding authors *These authors contributed equally to the work 1

2 Index of all supplementary tables, figures and notes Supplementary Tables Supplementary Table 1 Supplementary Table 2 Supplementary Table 3 Supplementary Table 4 Supplementary Table 5 Supplementary Table 6 Supplementary Table 7 Supplementary Table 8 Supplementary Table 9 Supplementary Table 10 Supplementary Table 11 Supplementary Table 12 Clinicopathological characteristics of 54 O. viverrini-associated CCA patients Sequence analysis summary of 8 O. viverrini-associated CCA exomes Non-synonymous somatic mutations identified and validated in the Discovery set of 8 tumors Somatic mutation spectra in O. viverrini-associated CCA Somatic mutations in TP53, KRAS, SMAD4, MLL3, GNAS, RNF43, ROBO2, RADIL, PEG3, NDC80, CDKN2A, PTEN, LAMA2, XIRP2 and PCDHA13 in 54 O. viverrini-associated CCA tumors Genome MuSiC analysis of mutated genes in O. viverrini-associated CCA TP53 mutations in O. viverrini-associated CCA Characteristics of O. viverrini-associated CCA patient with both SMAD4 and PTEN mutations Overall survival analysis in O. viverrini-associated CCA Mutation spectra in O. viverrini-associated Cholangiocarcinoma (CCA), Pancreatic cancer (PDAC) and Hepatocellular carcinoma (HCC) Chromosomal changes in Cholangiocarcinoma, Pancreatic cancer and Hepatocellular carcinoma Primers used for PCR amplification and Sanger sequencing Supplementary Figures Supplementary Figure 1 Distribution of mutations in 8 O. viverrini-associated CCA exomes Supplementary Figure 2 Summary of commonly mutated genes in 54 O. viverrini-associated CCA samples Supplementary Figure 3 Kaplan-Meier plots of overall survival of patients with tumors containing KRAS and RNF43 mutations Supplementary Figure 4 Analysis of LOH at ROBO2 by ASCAT (Allele-Specific Copy number Analysis of Tumors) in tumor W39, which also has an inactivating somatic mutation of ROBO2 Supplementary Figure 5 Comparisons of mutation spectra in O. viverrini-associated CCA, PDAC, and HCV-associated HCC Supplementary Notes Targeted exome capture and library preparation for massively parallel sequencing Supplementary References 2

3 Supplementary Table 1. Clinicopathological characteristics of 54 O. viverrini-associated CCA patients Code Sex Age TMN Staging Histological types Survival Status A028 F 59 T3N1M0 IVA WD tubular adenocarcinoma 474 DD A035 F 49 T3N0M0 III WD tubular adenocarcinoma 167 DD A039 F 72 T3N1M0 IVA PD tubular adenocarcinoma 874 AL A042 F 52 T3N1M0 IVA WD tubular adenocarcinoma 865 AL A043 M 45 T3N0M0 III Papillary carcinoma 426 DD A074 M 55 T4N0M0 IVA WD tubular adenocarcinoma 154 DD A105 M 53 T3N1M0 IVA WD tubular adenocarcinoma 592 DD A106 M 51 T3N1M0 IVA WD tubular adenocarcinoma 18 DD A107 M 54 T3N0M0 III WD tubular adenocarcinoma 302 DD A119 F 51 T3N0M0 III Papillary carcinoma 358 DD A120 F 53 T3N1M0 IVA Papillary carcinoma 156 DD A128 M 61 T3N1M0 IVA Papillary carcinoma 252 DD A142 F 38 T2bN1M0 IVA WD tubular adenocarcinoma 559 DD A159 M 55 T3N0M0 IIIB Papillary carcinoma 237 DD A162 M 68 T3N1M0 IVA Papillary carcinoma 617 AL B032 M 53 T3N0M0 III MD tubular adenocarcinoma 237 DD B048 F 51 T2bN0M0 IVA WD tubular adenocarcinoma 154 DD B070 M 61 T3N0M0 IIIB Papillary carcinoma 433 AL B083 F 53 T3N0M0 III WD tubular adenocarcinoma 266 DD B085 M 73 T3N0M0 III WD tubular adenocarcinoma 82 DD B087 M 56 T2bN0M0 II WD tubular adenocarcinoma 391 AL B099 M 48 T3N0M0 III WD tubular adenocarcinoma 373 AL B113 M 61 T3N1M0 IVA Papillary carcinoma 111 DD B149 M 63 T3N0M0 IIIA WD tubular adenocarcinoma 258 AL R100 F 49 T2bN1M0 IIIB WD tubular adenocarcinoma 251 DD R104 F 64 T3N0M0 III WD tubular adenocarcinoma 117 DD R134 M 50 T3N0M0 III WD tubular adenocarcinoma 222 DD R149 M 37 T4N1M0 IIIA Papillary carcinoma 475 DD T003 M 52 T4N0M0 IVA MD tubular adenocarcinoma 2253 DD T026 M 52 T3N1M0 IIIB WD tubular adenocarcinoma 307 DD 3

4 Supplementary Table 1 continued. Clinicopathological characteristics of 54 O. viverriniassociated CCA patients Code Sex Age TMN Staging Histological types Survival Status T151 M 73 T2bN1M1 IVB MD tubular adenocarcinoma 191 DD T157 M 42 T3N0M0 IIIa WD tubular adenocarcinoma 351 DD T160 M 46 T3N0M0 IIIa Papillary carcinoma 2451 AL U027 F 66 T2N1M0 IVA Papillary carcinoma 148 DD U044 M 57 T3N1M0 IIIB Papillary carcinoma 45 DD W012 F 61 T3N0M0 III WD tubular adenocarcinoma 149 DD W039 M 66 T3N0M0 III WD tubular adenocarcinoma 64 DD W040 M 56 T4N1M0 IVA WD tubular adenocarcinoma 157 DD Y002 F 45 T3N1M0 IVA Papillary carcinoma 1320 AL Y008 F 65 T3N0M0 III Papillary carcinoma 176 DD Y019 M 70 T4NXM0 IVA WD tubular adenocarcinoma 210 DD Y020 M 50 T3N1M0 IVA Papillary carcinoma 180 DD Y023 M 62 T4N0M0 IVA Papillary carcinoma 1274 AL Y032 M 69 T3N1M0 IVA Papillary carcinoma 429 DD Y033 F 51 T4N1M0 IVA WD tubular adenocarcinoma 157 DD Y035 F 64 T3N0M0 III WD tubular adenocarcinoma 754 DD Y057 M 57 T3N1M0 IVA WD tubular adenocarcinoma 416 DD Y065 M 63 T4N1M0 IVA Papillary carcinoma 558 DD Y072 M 61 T3N0M0 III Papillary carcinoma 302 DD Y074 F 40 T1N0M0 I Papillary carcinoma 1148 AL Y091 F 51 T4N0M0 IVA WD tubular adenocarcinoma 609 DD Y123 M 55 T4N0M0 IVA WD tubular adenocarcinoma 340 DD Y140 F 56 T2N0M0 II Papillary carcinoma 992 AL Y149 M 69 T4N1M0 IVA WD tubular adenocarcinoma 876 DD Highlighted in Bold = samples in Discovery set Histological types : tumor differentiation, WD = Well differentiated, MD = Moderately differentiated, PD = Poorly differentiated, PAP = Papillary Staging: TMN stage according to TMN classification, AJCC sixth edition. Survival : survival time in days. Status : follow up status ; DD = dead, AL = alive (data on 15 Aug 2011) 4

5 Supplementary Table 2. Sequence analysis summary of 8 O. viverrini-associated CCA exomes Bases in Target Region Bases Sequenced Bases Mapped to Genome * Bases After Filtering Bases Mapped to Target Region * Ave. Depth Per Targeted Base Targeted Bases with Depth at Least 1X Targeted Bases with Depth at Least 20X Somatic mutations identified in targeted region B085 B099 R104 T026 U044 W012 W039 W040 Normal 37,806,033 11,295,136,692 8,692,958,394 7,490,182,833 3,905,980, Tumor 37,806,033 11,255,244,354 8,756,261,352 7,496,830,908 3,812,200, Normal 37,806,033 7,097,490,664 5,960,436,264 5,199,247,030 2,930,117, Tumor 37,806,033 6,665,088,684 5,473,009,068 4,726,727,060 2,798,044, Normal 37,806,033 7,571,001,568 5,710,829,528 5,040,625,070 1,903,794, Tumor 37,806,033 7,418,858,456 5,981,689,212 5,293,556,256 2,668,373, Normal ,809,159,504 7,792,775,868 6,816,265,726 3,668,218, Tumor 37,806,033 8,949,919,640 7,988,945,428 6,972,740,798 3,782,109, Normal 37,806,033 7,264,404,768 6,099,026,424 5,395,414,370 2,526,282, Tumor 37,806,033 6,954,997,300 5,822,840,516 5,152,914,150 2,404,785, Normal 37,806,033 7,868,529,224 7,573,370,752 6,475,821,992 2,969,234, Tumor 37,806,033 6,755,744,456 6,473,749,104 5,451,167,598 2,519,928, Normal 37,806,033 6,349,013,668 6,118,662,324 5,167,650,066 2,428,397, Tumor 37,806,033 6,870,484,564 6,609,333,680 5,659,540,794 2,799,365, Normal 37,806,033 8,110,359,300 7,214,104,600 6,299,378,760 3,409,974, Tumor 37,806,033 8,848,149,808 7,906,602,224 6,911,891,116 3,673,858, AVERAGE 37,806,033 8,005,223,916 6,885,912,171 5,971,872,158 3,012,541, * based on the hg18 UCSC release of the human genome. 5

6 Supplementary Table 3. Non-synonymous somatic mutations identified and validated in the Discovery set of 8 tumors No. Gene Symbol Sample Transcript Accession ID Nucleotide (genomic) Nucleotide (cdna) Amino acid (protein) Mutation type 1 ADAM12 B085 CCDS g.chr10: C>T c.2104c>t p.r702w Missense 2 AFF1 W040 CCDS g.chr4: T>C c.2282t>c p.l761s Missense 3 ALPP B085 CCDS g.chr2: C>T c.814c>t p.r272c Missense 4 ANK2 T026 CCDS g.chr4: C>T c.3466c>t p.r1156c Missense 5 ANKRD35 U044 CCDS919.1 g.chr1: G>A c.2779g>a p.e927k Missense 6 ANO4 B085 CCDS g.chr12: G>T c.790g>t p.e264x Nonsense 7 ARAF U044 CCDS g.chrx: A>T c.650a>t p.n217i Missense 8 ARID1A T026 CCDS285.1 g.chr1: C>T c.2632c>t p.q878x Nonsense 9 ARL6IP5 U044 CCDS g.chr3: T>C c.317t>c p.m106t Missense 10 ASAP3 R104 CCDS235.1 g.chr1: G>A c.992g>a p.c331y Missense 11 BBS4 W040 CCDS10246 g.chr15: delGTT c.471 to 473 delgtt fs Deletion 12 BCAS2 R104 CCDS874 g.chr1: delC c.4 delg fs Deletion 13 CALML5 T026 CCDS g.chr10: C>T c.7c>t p.g3s Missense 14 CALN1 R104 CCDS g.chr7: A>G c.380a>g p.d127g Missense 15 CCDC97 R104 CCDS g.chr19: G>T c.730g>t p.e244x Nonsense 16 CD109 B099 CCDS g.chr6: C>T c.4240c>t p.r1414c Missense 17 CDH8 U044 CCDS g.chr16: G>A c.580g>a p.a194t Missense 18 CDKN2A U044 CCDS g.chr9: C>T c.416c>t p.r139q Missense 19 CHD5 R104 CCDS57.1 g.chr1: G>A c.4330g>a p.q1444x Nonsense 20 CHRD B085 CCDS g.chr3: G>A c.1513g>a p.v505m Missense 21 CNBP W040 CCDS g.chr3: C>T c.362c>t p.s121f Missense 22 COL11A1 W040 CCDS779.1 g.chr1: G>A c.793g>a p.e255k Missense 23 COL11A2 U044 CCDS g.chr6: C>T c.2842c>t p.r948c Missense 24 CRISPLD1 B085 CCDS g.chr8: G>A c.218g>a p.r73q Missense 25 CTNNA2 W040 CCDS g.chr2: T>A c.1748t>a p.m583k Missense 26 DDX52 B085 CCDS g.chr17: G>A c.1505g>a p.r502q Missense 27 DICER1 U044 CCDS g.chr14: G>A c.4651g>a p.e1551k Missense 28 DLGAP5 B099 CCDS g.chr14: G>C c.1150g>c p.g384r Missense 29 DSP U044 CCDS g.chr6: C>T c.4763c>t p.s1588f Missense 30 DYNC1H1 W039 CCDS g.chr14: G>A c.3182g>a p.w1061x Nonsense 31 EHBP1 R104 CCDS g.chr2: G>A c.47g>a p.s16f Missense 32 EIF2C1 B085 CCDS398.1 g.chr1: G>A c.1967g>a p.r656h Missense 33 EIF3E U044 CCDS g.chr8: C>G c.80c>g p.s27c Missense 34 ENTPD8 R104 CCDS g.chr9: C>T c.178c>t p.q60x Nonsense 35 EPHA2 R104 CCDS169.1 g.chr1: G>A IVS1+1 G>A Splice site Splice site 6

7 Supplementary Table 3 continued. Non-synonymous somatic mutations identified and validated in the Discovery set of 8 tumors No. Gene Symbol Sample Transcript Accession ID Nucleotide (genomic) Nucleotide (cdna) Amino acid (protein) Mutation type 36 EXOSC8 R104 CCDS g.chr13: G>A c.412g>a p.d138n Missense 37 EXTL3 W012 CCDS g.chr8: A>G c.2419a>g p.k807e Missense 38 FAM47A W040 CCDS g.chrx: G>A c.865g>a p.e289k Missense 39 FBLN1 W039 CCDS g.chr22: C>T c.1739c>t p.s580f Missense 40 FBN2 B099 CCDS g.chr5: T>A c.630t>a p.d210e Missense 41 FER W012 CCDS g.chr5: A>T c.973a>t p.n325y Missense 42 FH W039 CCDS g.chr1: C>T c.884c>t p.a295v Missense 43 FKBP3 T026 CCDS g.chr14: A>G c.235a>g p.s79g Missense 44 FLNC B085 CCDS g.chr7: G>A c.1037g>a p.g346e Missense 45 FMNL1 W039 CCDS g.chr17: G>A c.2755g>a p.v919m Missense 46 FOXR2 U044 ENST g.chrx: G>C c.775g>c p.d259h Missense 47 GATM U044 CCDS g.chr15: C>T c.565c>t p.r189c Missense 48 GDPD3 W040 CCDS10671 g.chr16: insAGCT c.938 insagct fs Insertion 49 GHRHR R104 CCDS5432 g.chr7: T>C c.346t>c p.s116p Missense 50 GHSR B085 CCDS g.chr3: G>A c.841g>a p.v281i Missense 51 GIGYF2 B099 CCDS g.chr2: G>A c.1136g>a p.r379t Missense 52 GJB6 W012 CCDS g.chr13: G>A c.380g>a p.r127q Missense 53 GLI3 T026 CCDS g.chr7: C>A c.664c>a p.l222m Missense 54 GNAS T026 CCDS g.chr20: C>T c.601c>t p.r201c Missense 55 GNAS W012 CCDS g.chr20: C>T c.601c>t p.r201c Missense 56 GPSM1 W039 CCDS48055 g.chr9: G>A c.441g>a p.r75q Missense 57 HCN4 B099 CCDS g.chr15: C>T c.1159c>t p.r387c Missense 58 HDAC2 W012 CCDS g.chr6: C>T c.514c>t p.r172w Missense 59 HDAC4 W040 CCDS g.chr2: C>T c.1633c>t p.p545s Missense 60 HECW2 R104 CCDS g.chr2: A>G c.1267a>g p.i423v Missense 61 HIST1H2AG U044 CCDS g.chr6: G>C c.364g>c p.e122q Missense 62 HOOK1 R104 CCDS612.1 g.chr1: G>T c.82g>t p.a28s Missense 63 HOXC11 U044 CCDS g.chr12: G>A c.454g>a p.d152n Missense 64 IL1RAPL1 T026 CCDS g.chrx: G>A c.277g>a p.g93r Missense 65 INHBA T026 CCDS g.chr7: C>T c.916c>t p.r306c Missense 66 IP6K2 R104 CCDS g.chr3: C>T c.37c>t p.r13c Missense 67 IQCB1 U044 CCDS g.chr3: G>T c.303g>t p.e101d Missense 68 IRX1 U044 CCDS g.chr5: G>C c.486>c p.m162i Missense 69 ITGA2B T026 CCDS g.chr17: G>A c.1708g>a p.g570r Missense 70 ITPR2 W039 CCDS g.chr12: C>T c.3766c>t p.l1256f Missense 7

8 Supplementary Table 3 continued. Non-synonymous somatic mutations identified and validated in the Discovery set of 8 tumors No. Gene Symbol Sample Transcript Accession ID Nucleotide (genomic) Nucleotide (cdna) Amino acid (protein) Mutation type 71 JAG1 B085 CCDS g.chr20: A>T c.2315a>t p.e772v Missense 72 KCNH5 W039 CCDS g.chr14: G>A c.989g>a p.r330q Missense 73 KCNH7 B085 CCDS g.chr2: G>A c.737g>a p.r246q Missense 74 KCNJ9 W040 CCDS1194 g.chr: insT c.241 inst fs Insertion 75 KCNN4 B085 CCDS g.chr19: C>T c.1163c>t p.s388l Missense 76 KCNQ2 R104 CCDS g.chr20: C>T c.1055c>t p.s352l Missense 77 KIAA1267 U044 CCDS g.chr17: G>A IVS4+5 G>A Splice site Splice site 78 KIAA1984 W040 CCDS43906 g.chr9: delCC c.504 to 505 delcc fs Deletion 79 KLHL4 W039 CCDS g.chrx: C>T c.646c>t p.l216f Missense 80 KRAS B085 CCDS g.chr12: G>C c.35g>c p.g12a Missense 81 KRAS R104 CCDS g.chr12: C>T c.358c>t p.g13d Missense 82 KRAS W012 CCDS g.chr12: G>C c.35g>c p.g12a Missense 83 LAMA2 U044 CCDS g.chr6: C>A c.289c>a p.h97n Missense 84 LAMA2 W039 CCDS g.chr6: T>A IVS14+5 T>A Splice site Splice site 85 LMX1A U044 CCDS g.chr1: G>C c.130g>c p.d44h Missense 86 LRP1B B085 CCDS g.chr2: A>G IVS69+6 A>G Splice site Splice site 87 LRP2 W012 CCDS g.chr2: G>A c.10652g>a p.r3551h Missense 88 LRRC15 R104 CCDS g.chr3: G>A c.1162g>a p.v388i Missense 89 LRRK2 W039 CCDS g.chr12: C>T c.575c>t p.s192l Missense 90 MAGEA11 B085 CCDS14690 g.chrx: G>T c.975g>t p.e325d Missense 91 MAP2K4 W039 CCDS g.chr17: T>G c.1043t>g p.l348r Missense 92 MAP3K13 R104 CCDS g.chr3: G>A c.801g>a p.m267i Missense 93 MARK4 R104 CCDS g.chr19: T>A c.320t>a p.v107d Missense 94 MASP2 B099 CCDS123.1 g.chr1: G>C c.2000g>c p.g667a Missense 95 MEFV W012 CCDS g.chr16: G>A c.2150g>a p.r717h Missense 96 MLL3 T026 CCDS g.chr7: G>T c.14641g>t p.e4881x Nonsense 97 MLL3 W039 CCDS g.chr7: G>T c.12149g>t p.s4050x Nonsense 98 MLYCD R104 CCDS g.chr16: G>C c.646g>c p.e216q Missense 99 MXRA5 U044 CCDS g.chrx: G>A c.3725g>a p.r1242q Missense 100 MYH2 W039 CCDS g.chr17: G>A c.1334g>a p.r445h Missense 101 NDC80 W012 CCDS g.chr18: delA c.756 dela fs Deletion 102 NDC80 W039 CCDS g.chr18: G>A c.859g>a p.e287k Missense 103 NFKB1 W040 CCDS43906 g.chr4: insC c.1032 insc fs Insertion 104 NFKBIL2 W040 CCDS g.chr8: C>T c.1567c>t p.p523s Missense 105 NLGN3 B085 CCDS g.chrx: G>A c.241g>a p.e81k Missense 8

9 Supplementary Table 3 continued. Non-synonymous somatic mutations identified and validated in the Discovery set of 8 tumors No. Gene Symbol Sample Transcript Accession ID Nucleotide (genomic) Nucleotide (cdna) Amino acid (protein) Mutation type 106 NPY5R U044 CCDS g.chr4: G>T c.250g>t p.a84s Missense 107 NUP160 R104 CCDS g.chr11: A>G c.996a>g p.k332n Missense 108 NUP160 R104 CCDS g.chr11: A>T c.994a>t p.k332e Missense 109 NUP210 W040 CCDS g.ch3r: G>A c.4054g>a p.e1352k Missense 110 ODF3 U044 CCDS g.chr11: T>G c.190t>g p.c64g Missense 111 OR51B2 T026 CCDS g.chr11: C>T c.83c>t p.p28l Missense 112 OR6Q1 R104 CCDS g.chr11: C>A c.662c>a p.s221y Missense 113 P2RY13 W012 CCDS g.chr3: C>T c.751c>t p.h251y Missense 114 P4HA3 B099 CCDS g.chr11: A>G c.377a>g p.d126g Missense 115 PAPD4 T026 CCDS g.chr5: C>T c.364c>t p.h122y Missense 116 PBX3 B099 CCDS g.chr9: A>T c.803a>t p.k268i Missense 117 PCBP3 U044 CCDS g.chr21: C>T c.512c>t p.p171l Nonsense 118 PCDH11X U044 CCDS g.chrx: G>A c.3616g>a p.a1206t Missense 119 PCDHA13 T026 CCDS g.chr5: C>T c.1985c>t p.t662m Missense 120 PCDHA13 U044 CCDS g.chr5: C>T c.1301c>t p.s434l Missense 121 PCDHA7 U044 CCDS g.chr5: G>A c.1354g>a p.a452t Missense 122 PCDHGA7 R104 ENST g.chr5: G>A c.2107g>a p.v703i Missense 123 PCNX W012 CCDS g.chr14: C>T c.4169c>t p.a1390v Missense 124 PDGFD W039 CCDS g.chr11: G>A c.970g>a p.v324m Missense 125 PEG3 W012 CCDS g.chr19: A>G c.2536a>g p.s846r Missense 126 PEG3 W039 CCDS g.chr19: G>A c.2947g>a p.d983n Missense 127 PGBD5 W039 CCDS g.chr1: C>T c.1139c>t p.t380m Missense 128 PHACTR4 B099 CCDS g.chr1: G>T c.1825g>t p.e609x Nonsense 129 POLDIP3 W040 CCDS14038 g.chr22: insC c.227 insc fs Insertion 130 POLL W039 CCDS g.chr10: G>A c.1157g>a p.r386h Missense 131 PPM1E T026 CCDS g.chr17: A>G c.1331a>g p.d444g Missense 132 PPP1R16B U044 CCDS g.chr20: A>G c.1675a>g p.k559e Missense 133 PPP2R3A W039 CCDS g.chr3: G>A c.37g>a p.d13n Missense 134 PRDM4 B099 CCDS g.chr12: T>C c.1117t>c p.f373l Missense 135 PREX2 W039 CCDS g.chr8: G>C c.4396g>c p.a1466p Missense 136 PRMT6 B085 CCDS g.chr1: A>G c.299a>g p.y100c Missense 137 PTEN R104 CCDS g.chr10: A>G c.464a>g p.y155c Missense 138 PTPRS W012 CCDS g.chr19: G>A c.230g>a p.r77h Missense 139 RAB11FIP5 R104 CCDS g.chr2: C>T c.1120c>t p.r374w Missense 140 RADIL W039 CCDS g.chr7: C>T IVS6+4 C>T Splice site Splice site 9

10 Supplementary Table 3 continued. Non-synonymous somatic mutations identified and validated in the Discovery set of 8 tumors No. Gene Symbol Sample Transcript Accession ID Nucleotide (genomic) Nucleotide (cdna) Amino acid (protein) Mutation type 141 RADIL W040 CCDS g.chr7: G>A c.386g>a p.r129q Missense 142 RASAL2 W012 CCDS g.chr1: C>T c.3776c>t p.t1259m Missense 143 RASGRF2 W039 CCDS g.chr5: A>G IVS2-2 A>G Splice site Splice site 144 RBBP7 W040 CCDS14179 g.chrx: delGGGTCATAACCA c.98 to 109 delgggtcataacca fs Deletion 145 RBM26 B099 CCDS g.chr13: G>C c.634g>c p.e212x Nonsense 146 RGS3 R104 CCDS g.chr9: C>T c.262c>t p.a88t Missense 147 RMI1 R104 CCDS g.chr9: A>G c.554a>g p.e185g Missense 148 RNF133 B099 CCDS g.chr7: G>T c.454g>t p.v152l Missense 149 RNF43 R104 CCDS g.chr17: A>G c.355a>g p.c119r Missense 150 RNF43 U044 CCDS g.chr17: A>T c.500a>t p.n167i Missense 151 RNF43 W012 CCDS g.chr17: C>T c.21667c>t p.q723x Nonsense 152 ROBO2 T026 CCDS g.chr3: C>T c.1585cc>t p.q529x Nonsense 153 ROBO2 W039 CCDS g.chr3: insC c.3234 insc fs Insertion 154 SACS B099 CCDS g.chr13: A>T c.5660a>t p.k1887i Missense 155 SAMD7 W039 CCDS g.chr3: C>G c.25c>g p.p9a Missense 156 SCN11A R104 CCDS g.chr3: C>T c.3470c>t p.a1157v Missense 157 SEMA4F W040 CCDS g.chr2: G>C c.2067g>c p.q689h Missense 158 SEMA5A B085 CCDS g.chr5: C>A c.2170c>a p.h724n Missense 159 SEMA6D T026 CCDS g.chr15: C>T IVS16-3 C>T Splice site Splice site 160 SGSM2 B099 CCDS g.chr17: G>C c.2045g>c p.s682t Missense 161 SIGLEC12 W012 CCDS g.chr19: C>T c.197c>t p.a66v Missense 162 SLC23A1 W040 CCDS g.chr5: A>T c.227a>t p.q76l Missense 163 SLC5A5 W040 CCDS g.chr19: G>A c.1682g>a p.g561e Missense 164 SMAD4 B099 CCDS g.chr18: T>C c.1283t>c p.k428t Missense 165 SMAD4 R104 CCDS g.chr18: delAGTA c.1447 to 1450 delagta fs Deletion 166 SMAD4 U044 CCDS g.chr18: C>T c.547c>t p.q183x Nonsense 167 SMAD4 W012 CCDS g.chr18: G>T c.394g>t p.h132d Missense 168 SMARCA1 B099 CCDS g.chrx: C>A c.2594c>a p.l532m Missense 169 SMC6 W040 CCDS g.chr2: A>G c.1658a>g p.y553c Missense 170 SNAPC4 B099 CCDS g.chr9: C>T c.443c>t p.p148l Missense 171 SPAG17 U044 CCDS899.1 g.chr1: G>A IVS1+3 G>A Splice site Splice site 172 SPSB4 B099 CCDS g.chr3: G>T c.592g>t p.g198c Missense 10

11 Supplementary Table 3 continued. Non-synonymous somatic mutations identified and validated in the Discovery set of 8 tumors No. Gene Symbol Sample Transcript Accession ID Nucleotide (genomic) Nucleotide (cdna) Amino acid (protein) Mutation type 173 STK36 T026 CCDS g.chr2: A>G c.2599a>g p.s867g Missense 174 STT3B R104 CCDS g.chr3: C>T c.398c>t p.p133l Missense 175 SYNE1 B099 CCDS g.chr6: C>T c.12953c>t p.t4318m Missense 176 SYNJ2 B085 CCDS g.chr6: C>A c.811c>a p.l271m Missense 177 TARS T026 CCDS g.chr5: C>T c.218c>t p.a73v Missense 178 TCEAL2 T026 CCDS g.chrx: C>T c.569c>t p.a190v Missense 179 TFAP2D B099 CCDS g.chr6: C>T c.1150c>t p.h384y Missense 180 THBS2 W039 CCDS g.chr6: C>T c.175c>t p.r59c Missense 181 TMCO4 W040 CCDS198.1 g.chr1: G>T c.327g>t p.l109f Missense 182 TMEM222 R104 CCDS297.2 g.chr1: G>A c.526g>a p.g176r Missense 183 TNKS2 W039 CCDS g.chr10: G>A c.2795g>a p.r932k Missense 184 TP53 T026 CCDS g.chr17: insG c.216 insg fs Insertion 185 TP53 U044 CCDS g.chr17: G>A c.310c>t p.q104x Nonsense 186 TP53 W012 CCDS g.chr17: G>A c.742g>a p.r248w Missense 187 TP53 W040 CCDS g.chr17: inst c.716 inst fs Insertion 188 TRIM28 B085 CCDS g.chr19: G>T c.1475g>t p.r492l Missense 189 TRIM48 W039 CCDS7947 g.chr11: C>T c.1048c>t p.q366x Nonsense 190 TROVE2 W040 CCDS1379 g.chr1: insTG c.1007 instg fs Insertion 191 TRPC5 B099 CCDS g.chrx: G>T c.1662g>t p.k554n Missense 192 TUBA3C U044 CCDS g.chr13: G>T c.1345g>t p.e449x Nonsense 193 UBAC1 W040 CCDS g.chr9: G>C c.484g>c p.v162m Missense 194 UTP20 W039 CCDS g.chr12: T>A IVS53+5 T>A Splice site Splice site 195 VANGL2 W040 CCDS g.chr1: T>A c.302t>a p.l101q Missense 196 VAV1 W040 CCDS g.chr19: G>A c.2383g>a p.a795t Missense 197 VCAN R104 CCDS g.chr5: G>A c.9904g>a p.v3302i Missense 198 VEGFC R104 CCDS g.chr4: A>G c.235a>g p.k79e Missense 199 VWCE T026 CCDS g.chr11: C>G c.732c>g p.f244l Missense 200 WNK3 B085 CCDS g.chrx: G>A c.5246g>a p.g1749e Missense 201 XIRP2 B085 CCDS g.chr2: inst c.9704 inst fs Insertion 202 XIRP2 T026 CCDS g.chr2: C>T c.7898c>t p.s2633l Missense 203 YSK4 W012 CCDS g.chr2: C>T c.812c>t p.s271l Missense 204 ZFY U044 CCDS g.chry: C>T c.1958c>t p.t653m Missense 205 ZNF136 W040 CCDS g.chr19: A>T c.569a>t p.h190l Missense 206 ZNF790 U044 CCDS g.chr19: G>A c.1840g>a p.e614k Missense 11

12 Supplementary Table 4. Somatic mutation spectra in O. viverrini-associated CCA Base pair change % Number Total number of substitutions Substitutions at C:G base pairs C:G to T:A C:G to G:C C:G to A:T Substitutions at T:A base pairs T:A to C:G T:A to G:C T:A to A:T Substitutions at specific dinucleotides 5'-CpG-3' '-TpC-3' NB: Mutations in this analysis include synonymous and non-synonymous coding mutations and mutations at canonical splice sites and in untranslated regions (Sanger validated and non-validated targets) for O. viverrini-associated CCA. 12

13 Supplementary Table 5. Somatic mutations in TP53, KRAS, SMAD4, MLL3, GNAS, RNF43, ROBO2, RADIL, PEG3, NDC80, CDKN2A, PTEN, LAMA2, XIRP2 and PCDHA13 in 54 O. viverrini-associated CCA tumors Sample Gene Symbol CCDS ID Nucleotide (genomic) Nucleotide (cdna) Amino acid (protein) Mutation type U044 CDKN2A CCDS g.chr9: C>T c.416c>t p.r139q Missense Y091 CDKN2A CCDS g.chr9: C>T c.238 C>T p.r80x Nonsense A106 CDKN2A CCDS g.chr9: G>T c.343 G>T p.v115l Missense A074 GNAS CCDS g.chr20: G>T c.602 G>T p.r201l Missense B149 GNAS CCDS g.chr20: C>T c.601 C>T p.r201c Missense T026 GNAS CCDS g.chr20: C>T c.601 C>T p.r201c Missense W012 GNAS CCDS g.chr20: C>T c.601 C>T p.r201c Missense Y149 GNAS CCDS g.chr20: G>A c.602 G>A p.r201h Missense B085 KRAS CCDS g.chr12: G>C c.35 G>C p.g12a Missense R104 KRAS CCDS g.chr12: C>T c.38 C>T p.g13d Missense T151 KRAS CCDS g.chr12: G>A c.35 G>A p.g12d Missense U027 KRAS CCDS g.chr12: G>A c.34 G>A p.g12s Missense W12 KRAS CCDS g.chr12: G>C c.35 G>C p.g12a Missense Y008 KRAS CCDS g.chr12: G>A c.35 G>A p.g12d Missense Y019 KRAS CCDS g.chr12: G>T c.34 G>T p.g12c Missense Y020 KRAS CCDS g.chr12: G>T c.35 G>T p.g12v Missense Y149 KRAS CCDS g.chr12: G>T c.35 G>T p.g12v Missense U044 LAMA2 CCDS g.chr6: C>A c.289c>a p.h97n Missense W039 LAMA2 CCDS g.chr6: T>A IVS14+5 T>A Splice site Splice site T026 MLL3 CCDS g.chr7: G>T c G>T p.e4881x Nonsense W039 MLL3 CCDS g.chr7: G>T c G>T p.s4050x Nonsense R100 MLL3 CCDS g.chr7: insTT c.11908_11909 ins TT fs Indel R149 MLL3 CCDS g.chr7: C>T c.9934 C>T p.q3312x Nonsense Y023 MLL3 CCDS g.chr7: G>A c G>A p.w4360x Nonsense Y057 MLL3 CCDS g.chr7: A>T c.411 A>T p.q147h Missense Y091 MLL3 CCDS g.chr7: T>C c T>C p.v4527a Missense B149 MLL3 CCDS g.chr7: C>A IVS2+2 C>A Splice site Splice site U027 MLL3 CCDS g.chr7: G>C c G>C p.l4331l Synonymous W012 NDC80 CCDS g.chr18: delA c.759 dela fs Deletion W039 NDC80 CCDS g.chr18: G>A c.859 G>A p.e287k Missense W012 PEG3 CCDS g.chr19: A>G c.2536 A>C p.s846r Missense W039 PEG3 CCDS g.chr19: G>A c.2947 G>A p.d983n Missense T157 PEG3 CCDS g.chr19: C>T c.2012 C>T p.s671f Missense A142 PEG3 CCDS g.chr19: C>T c.1215 C>T ph405h Synonymous 13

14 Supplementary Table 5 continued. Somatic mutations in TP53, KRAS, SMAD4, MLL3, GNAS, RNF43, ROBO2, RADIL, PEG3, NDC80, CDKN2A, PTEN, LAMA2, XIRP2 and PCDHA13 in 54 O. viverrini-associated CCA tumors Sample Gene Symbol CCDS ID Nucleotide (genomic) Nucleotide (cdna) Amino acid (protein) Mutation type T026 PCDHA13 CCDS g.chr5: C>T c.1985c>t p.t662m Missense U044 PCDHA13 CCDS g.chr5: C>T c.1301c>t p.s434l Missense R104 PTEN CCDS g.chr10: A>G c.464a>g p.y155c Missense A159 PTEN CCDS g.chr10: G>A c.509 G>A P.S170N Missense W040 RADIL CCDS g.chr7: G>A c.386 G>A p.r129q Missense W039 RADIL CCDS g.chr7: C>T IVS6+4 C>T Splice site Splice site A074 RNF43 CCDS g.chr17: C>G c.611 C>G p.t204r Missense A159 RNF43 CCDS g.chr17: C>T c.337 C>T p.r113x Nonsense R104 RNF43 CCDS g.chr17: A>G c.355 T>C p.c119r Missense U044 RNF43 CCDS g.chr17: A>T c.500 A>T p.n167i Missense W12 RNF43 CCDS g.chr17: C>T c.2167 C>T p.q723x Nonsense W039 ROBO2 CCDS g.chr3: insC c.3234 insc fs Insertion T026 ROBO2 CCDS g.chr3: C>T c.1585 C> T p.q529x Nonsense A159 ROBO2 CCDS g.chr3: T>A c.3999 T>A p.s1322r Missense Y020 ROBO2 CCDS g.chr3: G>T c.712 G>T p.e238x Nonsense T003 ROBO2 CCDS g.chr3: C>T c.2039 C>T p.a680v Missense A039 SMAD4 CCDS g.chr18: T>C c633 T>C p.t211t Synonymous A105 SMAD4 CCDS g.chr18: delga c.90 delga fs Deletion A159 SMAD4 CCDS g.chr18: G>A c.988 G>A p.e330k Missense A035 SMAD4 CCDS g.chr18: A>G c.1659 A>G p.x553w Missense B070 SMAD4 CCDS g.chr18: C>T c.346 C>T p.q116x Nonsense B099 SMAD4 CCDS g.chr18: T>C c.428 A>C p.k428t Missense B149 SMAD4 CCDS g.chr18: G>A c.404 G>A p.r135q Missense B149 SMAD4 CCDS g.chr18: C>T c.1081 C>T p.r361c Missense R104 SMAD4 CCDS g.chr18: delagta c.1447 delagta fs Deletion U044 SMAD4 CCDS g.chr18: C>T c.547 C>T p.q183x Nonsense W012 SMAD4 CCDS g.chr18: G>T c.394 C>G p.h132d Missense A042 TP53 CCDS g.chr17: delt c.582 delt fs Deletion A105 TP53 CCDS g.chr17: T>G c.981 T>G p.y327x Nonsense A107 TP53 CCDS g.chr17: A>G c.578 A>G p.h193r Missense A119 TP53 CCDS g.chr17: C>T c.844 C>T p.r282w Missense A120 TP53 CCDS g.chr17: G>A c.818 G>A p.r273h Missense A142 TP53 CCDS g.chr17: G>A c.673 G>A p.v225i Missense A159 TP53 CCDS g.chr17: C>T c.916 C>T p.r306x Nonsense A162 TP53 CCDS g.chr17: A>G c.395 A>G p.k132r Missense B032 TP53 CCDS g.chr17: C>G c.832 C>G p.p278s Missense 14

15 Supplementary Table 5 continued. Somatic mutations in TP53, KRAS, SMAD4, MLL3, GNAS, RNF43, ROBO2, RADIL, PEG3, NDC80, CDKN2A, PTEN, LAMA2, XIRP2 and PCDHA13 in 54 O. viverrini-associated CCA tumors Sample Gene Symbol CCDS ID Nucleotide (genomic) Nucleotide (cdna) Amino acid (protein) Mutation type B070 TP53 CCDS g.chr17: C>T c.707 C>T p.y236c Missense B083 TP53 CCDS g.chr17: T>C c.403 T>C p.c135r Missense B099 TP53 CCDS g.chr17: G>T & inst c.686 G>T & fs Insertion ins T R134 TP53 CCDS g.chr17: G>T c.1027 G>T p.e343x Nonsense R149 TP53 CCDS g.chr17: G>A c. 853 G>A p.e285k Missense T157 TP53 CCDS g.chr17: G>A c.375 G>A p.t125t Synonymous T026 TP53 CCDS g.chr17: insG c.216 insg fs Insertion U044 TP53 CCDS g.chr17: G>A c.310 C>T p.q104x Nonsense W012 TP53 CCDS g.chr17: G>A c.742 C>T p.r248w Missense W040 TP53 CCDS g.chr17: insT c.716 ins T fs Insertion Y002 TP53 CCDS g.chr17: C>T c.586 C>T p.r196x Nonsense Y008 TP53 CCDS g.chr17: G>A c.644 G>A p.s215n Missense Y020 TP53 CCDS g.chr17: T>G c.489 T>G p.y163x Nonsense Y035 TP53 CCDS g.chr17: C>T c.817 C>T p.r273c Missense Y072 TP53 CCDS g. chr17: del c. del 750bp fs Deletion Y149 TP53 CCDS g.chr17: C>T c.817 C>T p.r273h Missense B085 XIRP2 CCDS g.chr2: inst c.9704 inst fs Insertion T026 XIRP2 CCDS g.chr2: C>T c.7898c>t p.s2633l Missense T160 XIRP2 CCDS g.chr2: C>T c.218 C>T p.s73l Missense Bold = Somatic mutation identified in discovery screening. 15

16 Supplementary Table 6. Genome MuSiC analysis of mutated genes in O. viverrini-associated CCA a) Ranking of genes according to probabilities that they harbor more non-synonymous mutations than expected by chance given the background mutation rate of the discovery set. Likelihood Ratio Test Convolution Test Rank Gene SNVs Indels P-value LRT FDR LRT P-value CT FDR CT 1 TP E E E E-06 2 SMAD E E E-05 3 KRAS E E RNF E E NDC E E GNAS E = ROBO = RPL10L = TAPBP = UFSP = PEG = XIRP = PCDHA = MLL = PTEN = CDKN2A LAMA = DNAH = RADIL We used the longest CCDS transcript (or, if no CCDS transcript was available, the longest RefSeq transcript). Rankings were computed by Genome MuSiC ( and genes are sorted by the Convolution Test FDR and then the Likelihood Ratio Test FDR. The 15 genes validated in this study (Table 1) are highlighted in orange. 16

17 b) Ranking of genes according to probabilities that they harbor more non-synonymous mutations than expected by chance given the background mutation rate, based on mutations detected in both the discovery and prevalence sets. Likelihood Ratio Test Convolution Test Rank Gene SNVs Indels P FDR P FDR 1 KRAS SMAD TP GNAS E E E E-11 5 MLL E E E E-11 6 RNF E E E E-10 7 ROBO E E E E-09 8 CDKN2A E E E E-07 9 PEG E E E E XIRP E E E E NDC E E E E PTEN E E E PCDHA LAMA RADIL c) Ranking of genes according to probabilities that they harbor more non-synonymous mutations than expected by chance given the background mutation rate, based on mutations detected in the prevalence set only. Likelihood Ratio Test Convolution Test Rank Gene SNVs Indels P-value FDR P-value FDR 1 TP KRAS E E-18 3 SMAD E E E E-14 4 GNAS E E E E-06 5 MLL E E E E-06 6 CDKN2A E E E E-05 7 ROBO E E-05 8 RNF PTEN PEG XIRP LAMA NDC PCDHA RADIL

18 Supplementary Table 7: TP53 mutations in O. viverrini-associated CCA Sample Gene Symbol CCDS ID Nucleotide (genomic) Nucleotide (cdna) Amino acid (protein) Mutation type A042 TP53 CCDS g.chr17: delt c.582 delt fs (194) Deletion Y072 TP53 CCDS B099 TP53 CCDS g. chr17: del g.chr17: G>T & inst c. del 750bp fs (250) Deletion c.686 G>T & ins T fs (228) Insertion P53 gene structureeffected domain Core domain/specific DNA binding domain ( ) Core domain/specific DNA binding domain ( ) Core domain/specific DNA binding domain ( ) T026 TP53 CCDS g.chr17: insG c.216 insg fs (72) Insertion Yes W040 TP53 CCDS g.chr17: inst c.716 ins T fs (238) Insertion Core domain/specific DNA binding domain ( ) A105 TP53 CCDS g.chr17: T>G c.981 T>G p.y327x Nonsense Tetramerization ( ) Yes A159 TP53 CCDS g.chr17: C>T c.916 C>T p.r306x Nonsense Yes R134 TP53 CCDS g.chr17: G>T c.1027 G>T p.e343x Nonsense Tetramerization ( ) Yes U044 TP53 CCDS g.chr17: G>A c.310 C>T p.q104x Nonsense Yes COSMIC Yes Yes Yes Yes Y002 TP53 CCDS g.chr17: C>T c.586 C>T p.r196x Nonsense Y020 TP53 CCDS g.chr17: T>G c.489 T>G p.y163x Nonsense A107 TP53 CCDS g.chr17: A>G c.578 A>G p.h193r Missense A119 TP53 CCDS g.chr17: C>T c.844 C>T p.r282w* Missense A120 TP53 CCDS g.chr17: G>A c.818 G>A p.r273h* Missense A142 TP53 CCDS g.chr17: G>A c.673 G>A p.v225i Missense A162 TP53 CCDS g.chr17: A>G c.395 A>G p.k132r Missense B032 TP53 CCDS g.chr17: C>G c.832 C>G p.p278s* Missense B070 TP53 CCDS g.chr17: C>T c.707 C>T p.y236c Missense B083 TP53 CCDS g.chr17: T>C c.403 T>C p.c135r Missense R149 TP53 CCDS g.chr17: G>A c. 853 G>A p.e285k Missense W012 TP53 CCDS g.chr17: G>A c.742 C>T p.r248w* Missense Y008 TP53 CCDS g.chr17: G>A c.644 G>A p.s215n Missense Y035 TP53 CCDS g.chr17: C>T c.817 C>T p.r273c* Missense Y149 TP53 CCDS g.chr17: C>T c.817 C>T p.r273h* Missense T157 TP53 CCDS g.chr17: G>A c.375 G>A p.t125t Synonymous Core domain/specific DNA binding domain ( ) Core domain/specific DNA binding domain ( ) Core domain/specific DNA binding domain ( ) Core domain/specific DNA binding domain ( ) Core domain/specific DNA binding domain ( ) Core domain/specific DNA binding domain ( ) Core domain/specific DNA binding domain ( ) Core domain/specific DNA binding domain ( ) Core domain/specific DNA binding domain ( ) Core domain/specific DNA binding domain ( ) Core domain/specific DNA binding domain ( ) Core domain/specific DNA binding domain ( ) Core domain/specific DNA binding domain ( ) Core domain/specific DNA binding domain ( ) Core domain/specific DNA binding domain ( ) Core domain/specific DNA binding domain ( ) Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Transactivation domain (1-50), Core domain/specific DNA binding domain ( ), Tetramerization ( ), Regulatory domain ( ) Reference: Monique G. C. T. van Oijen and Pieter J. Slootweg (2000) 1 * Represent reported TP53 hot spot mutation 18

19 Supplementary Table 8. Characteristics of O. viverrini-associated CCA patient with both SMAD4 and PTEN mutations Sample Age Sex Histology Stage SMAD4 mutation PTEN mutation R104 F 64 WD tubular adenocarcinoma III Frameshift Missense A159 M 55 Papillary carcinoma III Missense Missense 19

20 Supplementary Table 9: Overall survival analysis in O. viverrini-associated CCA a) Influencing mutation factors on overall survival in O. viverrini-associated CCA (n=53) Independent Variable Patient Number Univariate analysis* (P-value) TP53 mutation Absent 29 Present KRAS mutation Absent 44 Present SMAD4 mutation Absent 44 Present MLL3 mutation Absent 45 Present ROBO2 mutation Absent 48 Present GNAS mutation Absent 48 Present RNF43 mutation Absent 48 Present 5 <0.001 * Significant at a Bonferroni corrected alpha of (0.05 / 7). 20

21 b) Multivariate Cox Proportional Hazard Model for prognostic factors in O. viverriniassociated CCA patients Independent Variable Age (<56 vs >56) Sex (Female vs Male) Histology type (Papillary vs Non papillary type) Univariate Multivariate Hazard ratio analysis analysis p value* p value** (95% CI) TNM stage (I /II /III vs IIIA,B,C /IVA,B) RNF43 mutation (Absent vs Present) <0.001 < ( ) Independent Variable Univariate analysis Multivariate analysis Age (<56 vs >56) Sex (Female vs Male) Histology type (Papillary vs Non papillary type) Hazard ratio p value* p value** (95% CI) TNM stage (I /II /III vs IIIA,B,C /IVA,B) KRAS mutation (Absent vs Present) ( ) *P value < 0.05 from log rank test **P value < 0.05 from Multivariate Cox proportional hazard model analysis NB: TNM stage did not show prognostic significance because only 3 cases are of stage I and II, while the rest of the cases are of late stage disease (III and IV). 21

22 c) Multivariate Cox Proportional Hazard Model for prognostic factors in O. viverriniassociated CCA patients (Excluding 2 cases harboring both KRAS and RNF43 mutations) Univariate Multivariate Hazard ratio Independent Variable analysis analysis p value* p value** (95% CI) Age (<56 vs >56) Sex (Female vs Male) Histology type (Papillary vs Non papillary type) TNM stage (I /II /III vs IIIA,B,C /IVA,B) RNF43 mutation (Absent vs Present) ( ) Univariate Multivariate Hazard ratio Independent Variable analysis analysis p value* p value** (95% CI) Age (<56 vs >56) Sex (Female vs Male) Histology type (Papillary vs Non papillary type) TNM stage (I /II /III vs IIIA,B,C /IVA,B) KRAS mutation (Absent vs Present) *P value < 0.05 from log rank test **P value < 0.05 from Multivariate Cox proportional hazard model analysis NB: TNM stage did not show prognostic significance because only 3 cases are of stage I and II, while the rest of the cases are of late stage disease (III and IV). 22

23 d) Multivariate Cox Proportional Hazard Model for prognostic factors including RNF43 and KRAS mutations as independent variable in O. viverrini-associated CCA patients Independent Variable Age (<56 vs >56) Sex (Female vs Male) Histology type (Papillary vs Non papillary type) Univariate Multivariate Hazard ratio analysis analysis p value* p value** (95% CI) TNM stage (I /II /III vs IIIA,B,C /IVA,B) RNF43 mutation (Absent vs Present) ( ) KRAS mutation (Absent vs Present) ( ) *P value < 0.05 from log rank test **P value < 0.05 from Multivariate Cox proportional hazard model analysis NB: TNM stage did not show prognostic significance because only 3 cases are of stage I and II, while the rest of the cases are of late stage disease (III and IV). 23

24 Supplementary Table 10. Mutation spectra in O. viverrini-associated Cholangiocarcinoma (CCA), Pancreatic cancer (PDAC) and Hepatocellular carcinoma (HCC) Mutation Category O. viverrini-associated Cholangiocarcinoma (CCA) Pancreatic ductal adenocarcinoma (PDAC)* HCV-associated Hepatocellular carcinoma (HCC) # CpG -> TpG TpC -> Tp* (not in TpCpG -> TpTpG) C:G->T:A (other than above) C:G->G:C (other than above) C:G->A:T (other than above) T:A->A:T T:A->G:C T:A->C:G Total P-values (ChiSq) $ E-16 * Data from Jones et al., Science 321, , 2008 # Data from Li et al., Nature Genetics 43, , 2011 $ P values for pairwise comparison of mutation spectrum of CCA with PDAC or HCC NB: Nine mutations in UTRs were excluded from PDAC counts. CCA mutations in this analysis include synonymous and non-synonymous coding mutations and mutations at canonical splice sites and in UTRs (Sanger validated and non-validated targets) for O. viverrini-associated CCA. 24

25 Supplementary Table 11. Chromosomal changes in Cholangiocarcinoma, Pancreatic cancer and Hepatocellular carcinoma Chromosome Chr1 Cholangiocarcinoma (CCA) 2,3,4 Pancreatic cancer (PDAC) 5,6 Hepatocellularcarcinoma (HCC) 7,8 +1q +1q +1q -1p -1p -1p Chr2 2 Chr3 +3q 3-3p -3p Chr4-4q Chr5 +5p +5 q and p Chr6-6q - 6 q and p Chr7 +7q and p +7p Chr8 +6p +8q +8q +8q -8p -8p Chr9-9p -9p Chr10 Chr11 Chr q and p Chr13-13q -13q -13q Chr14-14q Chr15 +14q Chr16-16q Chr17-17p -14q Chr18-18q -18q Chr19-6q -14q +17q +17q -17p -17p Chr20 +20q +20q Chr21 Chr22 Chr X Chr Y +Xq Yellow: Common between CCA and PDAC Green: Common between CCA and HCC Gray: Common to all 3 cancers 25

26 Supplementary Table 12. Primers used for PCR amplification and Sanger sequencing Gene Symbol Transcript IDs Coding Exon Number Genomic Region of Interest Forward primer sequence Reverse primer sequence Additional primer sequence for sequencing CDKN2A ENST Exon1 Chr9: _ GGTCCCTCCAGAGGATTTGAG GATTACAAACCCCTTCTGAAAAC CDKN2A ENST Exon2 Chr9: _ GAATGGATAGAGAACTCAAGAAGG GTGGTGGTATGCTTTGGGAAG CDKN2A ENST Exon3 Chr9: _ CCATTGCGAGAACTTTATCCATAAG CTCCAGAAAACTCCAACACAGTG GNAS ENST Exon8 and 9 Chr20: _ CAGCTTCCTGGACAAGATCGACGTG CTTGAAGAGGTTCAGAGCCTCCTGC KRAS ENST Exon2 Chr12: _ ACACGTCTGCAGTCAACTGG CATATAACTTGAAACCCAAGGTAC KRAS ENST Exon3 Chr12: _ CCTGACTATTGATGATGTTGAG CAAGTTACTCCACTGCTCTAATC MLL3 ENST Exon1 chr7: _ GAAATGCGAGAGGCTGAGC GCTCCCTCGCACTCAACTTAC GGATCCATGTGTCTTTCCTGG (F) MLL3 ENST Exon2 chr7: _ GGAGAAAATGACTGTAAACTCTG * CAAACACTAGATGGTTCATAGGCCAG MLL3 ENST Exon3 chr7: _ CGTGCATGAACTTTTCTTGTG CTAAACACAGAGGTATCAGCTTC MLL3 ENST Exon4 chr7: _ CTTATGGGTAATCACTGAATAGACTG ATTGGGTTAATGGTGAGTCAGAG MLL3 ENST Exon5 chr7: _ ACTTCCCTGTGGGTTTGGAGACTC GCATCCAGTCCGGTGGTGGTACTG CAAGGCAGATGGGAAACTTAGAGTTC [R] MLL3 ENST Exon6 chr7: _ GAAGGGCCACTTTACTGCTATCAC * CCAGATTCAAGCAAATTCTCCTG-PCR ACCATACCTGGCCTAGAATTTCTC [R] MLL3 ENST Exon7 chr7: _ CAGCGATGTGCATTTTGTAAGCACC CGATCCCTGGCAACGATCCCTCAG MLL3 ENST Exon8 chr7: _ GTCTGAAAGGTTCATGATGCAAATCC TGATGAAGTCACTGAATGAATATAGC MLL3 ENST Exon9 chr7: _ GATCAGAGGCTATTCATCTTGC GAGGGTGATGACTATGTTCATTATC CAGGGTTACCTCTGTTACTATCTGC (F) MLL3 ENST Exon10 chr7: _ GCAACAGAGTAAGAGTCCATCTC * CCTATTAGTGCTCATTTCTGATGTGG CCATCTATAACAGCAATCAGTAC [R] MLL3 ENST Exon11 chr7: _ CAACTCCTCTAACCTCTCTCCTCTC * CCATCAGATTCAGAGATGACCAAG CAGCTTTTCAGCAATGCCTTC (F) MLL3 ENST Exon12 chr7: _ CAGGCATGAACTACCACGGTTGG GATGGTAGCTGAACTACAAATACACG MLL3 ENST Exon13 chr7: _ CTTACTAATAAGTGGTACAATGTTTCATGC CCTTTCAAGAGAAACTAACCTTC MLL3 ENST Exon14-1 GTGGCTGATTCTAATCCATAAATCC GAGTCATTTCAGAGTCCATCAATCC chr7: _ MLL3 ENST Exon14-2 CAGTTGTTAGAGGAACCTGAAACAG CATTACACTGAAGTTCTATCTGAAGAC MLL3 ENST Exon15 chr7: _ GAGTCAATGTTGATGTCCTATAAACC GTGTTTCTTGGAAGCTCAATCTAG MLL3 ENST Exon16 chr7: _ CCAGGTCTCTTAATACTTATCTAAGG GGACAGTAAAATAAACATTTTGACC MLL3 ENST Exon17 chr7: _ GTCACGACTCCATTTTACGGTAG GTAATCTTCCTTCTGCTCCTTGG MLL3 ENST Exon18 chr7: _ CCTCATTGAATTCCTCCTTGCAC GTGAGCATAGAGGAGAGAGAATGC MLL3 ENST Exon19 chr7: _ GCTTCTAGGTACATAAGCACCAT GCAACTGACCCAGGAAGACTCC MLL3 ENST Exon20 chr7: _ GTTATCACACCTACTGCCTAGACC * CAAAACAGGAAATCGCTTCTGTAAG CAGAGCTTTCTCATACCACTTTGG (F) MLL3 ENST Exon21 chr7: _ CCTCTCTAATAGTTGTTCTCATTCC CAAGTTGTGATAAGTCATGTATAACC MLL3 ENST Exon22 chr7: _ TCCTCCATGACTTGAGTATTTCC * GCTAACTAGGCCGTGTTTTCTGC GTGGAAATGATTACCAACATTACAG [R] MLL3 ENST Exon23 chr7: _ CATGCAACCTTTCTGCCTGTAATCC ATGGCAAGGGTCAGTCACATCAG MLL3 ENST Exon24 chr7: _ GGAACACTGCACTAAGCAGCAC GGTTAACATTTGGGACAAGGCATTC MLL3 ENST Exon25 chr7: _ CAGGTTATTGATTTAGATTAGCACAG * GTAACAATAAGAGGCAATAAGGCTC GCAGTTTTATCCTGTGTACTGTGG (F) MLL3 ENST Exon26 chr7: _ AGTTGCCATTTCGTCATACTCATG ATGGCATTTATGATGACTAAATGAC 26

27 Supplementary Table 12 continued. Primers used for PCR amplification and Sanger sequencing Gene Symbol Transcript IDs Coding Exon Number Genomic Region of Interest Forward primer sequence Reverse primer sequence Additional primer sequence for sequencing MLL3 ENST Exon27 chr7: CTAGAAACCTTTGGTATAATTGTGTGG GCTACATCTTATACACCTGTCCAC MLL3 ENST Exon28 chr7: _ CATTAGTCACAAGACAAAATCGTCTG ATGCTGAGTCCTTTAACAAAGC MLL3 ENST Exon29 and30 chr7: _ CTGAGCGACTCTCCAAATTGATATG GCTGAGTGTTCGCAGGACTAAG MLL3 ENST Exon31 chr7: _ TATTCTAGGAGCAATTCTTGGA * CTCCCTATCAGTTTGTATACGTGG CCACTGGACATAAGGATAATTACAG [R] MLL3 ENST Exon32 chr7: _ AGTTGGTCCTCTCTATGCATGG GCAAAAGCCACAATAAACGCAA MLL3 ENST Exon33 chr7: _ GTTGCGTTTATTGTGGCTTTTGC * GAATCACATAACACGGATTTCTGC GGAATCTTCATGTTGTGGGTCATC [R] MLL3 ENST Exon34 chr7: _ CAGTGTGCCACGATCTTTGTTC CCAACTGTACCTCTGCTGAAGAG MLL3 ENST Exon35 chr7: _ GAGGTGCCGTAAGTATTTCAGC CTCATCATGGTATGTTCCATTTGTG MLL3 ENST Exon36-1 CTAATATGGGAGCAATCTGGTAGC GATGGCCTATTTGCTGTTGTCTC MLL3 ENST Exon36-2 CAGGATAGTCTTTCTCAGGCTCA ATAGGTCTTGGTGTCAAAGCAGG MLL3 ENST Exon36-3 chr7: _ CCTGTAGTTTCAGAACAAACTGC GAAATTCCAGGTCTTGGTGTTCC MLL3 ENST Exon36-4 CCAACATCTCAGGACCCATACTC CAGGAAGTTGGGAGACACCAGAG MLL3 ENST Exon36-5 GTTGGTAAGGCCACCTGATACATG CCAGACAACTATCACAAAACAAAGC MLL3 ENST Exon37 chr7: _ CTTTGAAACATTACCATAGCCAC GCAGTAACAGCTTCCTGATTAATAC MLL3 ENST Exon38-1 GCTCAGATATGGTGTTCATTATAGG * CCTCTAGACCATCAGAAGGCTGG GACAAAGGAGCTTCTGAAAATTCAC [R] MLL3 ENST Exon38-2 CTGAGACATGGAAACTTCATTCC GTGAGCAAGGAGTGTCAACATTATC CCTAATCAGCTACCTGTGCACC (F) chr7: _ MLL3 ENST Exon38-3 CAGAACTTGACATGGGAGATAAG CTGTTGATTGACCTGGAATGAGC MLL3 ENST Exon38-4 GGATCAACTCCAGTTCTCTCAAG CAAACTCCTCAAGGAGGAATAG MLL3 ENST Exon39 chr7: _ CCAGGATAAGGTTGCCATCTCAG GATCATATCGAGCTGATGCCATGA MLL3 ENST Exon40 chr7: _ GTACATGAAAGGTAGGGCTTTAGTG GATAAGGCTACTATTTAGGTTGGTG MLL3 ENST Exon41 chr7: _ GTTGCCACCACTAAATTCACTCTAC * GTGTCCTTGTCACTTAGGAAGACAG CAGTGATGTTTAATAAGACAGGAGG [R] MLL3 ENST Exon42 chr7: _ CAGTATCTCAAGAATGTGCATTCTC ACTAAGAGGTCTCAGAGCTGTCAC MLL3 ENST Exon43-1 GTGACTTTTGCAGTTACCCAAGC GTCGGAACTGTAGAAGGGAATCTG MLL3 ENST Exon43-2 CAAATGCAAATCCACAGAGTGG GATCCCGGATAACTGTGTCCATG chr7: _ MLL3 ENST Exon43-3 GACTAATGAGCGAAGGCAGGTAG GACTCTGTCTCAGCCTTTTCCAG MLL3 ENST Exon43-4 CTCCAGGCATCTCAGAAACTACC GGGTTCCAGAGAGGTAGAAATGAC MLL3 ENST Exon44 chr7: _ GCATTGACACCACTCTATCTTCC CTAGAACTCATGCCTCAAGTGATC MLL3 ENST Exon45 and 46 chr7: _ GGTGTGCAAGATACATCTTGATTAC * CCTGAAGATTATTCACCAAATTGTAC CATGAAGGTATGATTATAGCCTCC (F) MLL3 ENST Exon47 and 48 chr7: _ CATCGTATAGGTGCTGCCCAGAG GCTCTGCTCTCTGACCCTTTATCC MLL3 ENST Exon49 chr7: _ GGAATCCCTTTATGAGGATTTCTG GGTAAGTTCACCAAGGACTATACG MLL3 ENST Exon50 chr7: _ GATAATCGACTCTGTAACCATTGC * CCTTTAACAGCCTGTTGTAGCTC CAGGAACATGTGTCCATCACAC [R] MLL3 ENST Exon51 chr7: _ GAGTGAACCGTGGACCACATAG AGAGTAGCACGACTCAAGGCTCC 27

28 Supplementary Table 12 continued. Primers used for PCR amplification and Sanger sequencing Gene Symbol Transcript IDs Coding Exon Number Genomic Region of Interest Forward primer sequence Reverse primer sequence MLL3 ENST Exon53 chr7: _ GCTGCCATTTAATAAACAGGTTC CCAATGCCTTATTCCTCTAAGAG MLL3 ENST Exon54 chr7: _ CCTAAGTTTGGCTTTGAAACTG GTTCTCTTATGTAGTTGGGCACATAC MLL3 ENST Exon55 chr7: _ CTGCTAGGCAGCTTTCCATGTATG CCAAGGGACAGGATTTGATACG MLL3 ENST Exon56 chr7: _ GTAGGAATAAGCTAGTGATGGCATAG GGAATCAGTAGGTTGTGTGTGAAGC Additional primer sequence for sequencing MLL3 ENST Exon57 and 58 chr7: _ CGTGCCATGTAAATGTCATCTGTG * CATCTGGCCTATTATGGACAGAG GCTGAATGCAGCATGACAGATTTC [R] MLL3 ENST Exon59 chr7: _ GAACCTCAATAAATCAGGTCTTCC CTGCTTTAACCTAAAGGACTGAGG NDC80 ENST Exon2 chr18: _ CCTTGCAAATGAGAACTTCAGTG CAACACCTAGCAGAGTATCATGC NDC80 ENST Exon3 chr18: _ CTTATAGTACCATGTTTAGCCCATCC * CTCCACCGTGTCTGAAAGCTAGG GTGTTAGCTACACTGTCTACAGCAG [R] NDC80 ENST Exon4 and 5 chr18: _ CGTTATTTGCTCCTTTTCTCTGC TCCTACATATATTGCCCACAGTGG NDC80 ENST Exon6 chr18: _ GTATAGGTCAAGTTCTGCAGCTTCG CACTGTATTCTCCAAACTTCTTGC NDC80 ENST Exon7 chr18: _ GAGTCCATCCTTTCTAGATCCTGAAG CTATGACGTTTGGCAGGTTAAGTG NDC80 ENST Exon8 chr18: _ ATAAGCTTCCCTGTTTGTTTGACC ACTTCTCTCCACCAAAGAAACCAC NDC80 ENST Exon9 chr18: _ GAAGTACGGTGGAGTAATGGAAAG ACAAGGCTCCACAGATACCAAAC NDC80 ENST Exon10 chr18: _ GTGAAGCACTCAAACTGTTGTTG CAAACTTGTGATCATTTGTGACAGC NDC80 ENST Exon11 chr18: _ CCATTGCAAGCTAATAGGGAAATAAG * GGACTCATTTCCTCTTCTACAGAG CTGAATTGACATAAAGGGATGAGTG [R] NDC80 ENST Exon12 chr18: _ GGACTCTTATTCCAAGCTGTCAG GCAACTTGACAGTAAGGGTTAGG NDC80 ENST Exon13 chr18: _ GCAATGAATTTCCAGGATAGATGAG TGTCGAGAGCATACCCAATTCTC NDC80 ENST Exon14 chr18: _ GGACAAATGATAAGTGAGAGTCC GCAGAGAAAACTAACCCAAGATAC NDC80 ENST Exon15 chr18: _ CATAGTTGGAATTACAGGCACACACC GTATCTGTTGATAGGGAAAGTTCTCG NDC80 ENST Exon16 chr18: _ GTTACAACCCTTCGCTCCAAAGC GTTGCCACATCACTAGACAAGATTC NDC80 ENST Exon17 chr18: _ CCTCTTGCATAGTTGGAATTACAGG CAACCTGTATTTTGCCGACTTCTGG PEG3 ENST Exon4 chr19: _ CATTGTCGATGCCTCTGGTAAGG CAGCTCAATATCCCACAGTACACAGG PEG3 ENST Exon5 chr19: _ GTATGCCAGGAGTGGTTGGAAGC CTCAGATCTCTGAGTGAAGCTGACC PEG3 ENST Exon6 chr19: _ GAGCTTTGGAACTTGCTTGGAAG CTGGTGGTACCGAGTGGAACTTC PEG3 ENST Exon7 chr19: _ CTGCCACAGAATGCAGAGAAGAG GCTACTTGTCACCTAAGGGATGG PEG3 ENST Exon8 chr19: _ CGTAGGTCCCTAGTAACACTTGAC GTATCTTTACCCAAAGCCAGAGG PEG3 ENST Exon9 chr19: _ GTCTTCTGACATCCAAGGAATCAG CCAAATGTAAATACTCCCTAGTCC PEG3 ENST Exon10-1 CAACCAGTGAGATGCTATCAAGG CAATTGGCTGTGACTCGGTAAAG PEG3 ENST Exon10-2 CGCTATCATTTTGACACAGATGG TCACGTTCACGTTCATGTTCAC PEG3 ENST Exon10-3 GGCAAAGACAAATTCTACGAGTG TCGTCCTCATCACTTTCAAGAGG PEG3 ENST Exon10-4 AAGCTCAGAGCTCAGTGAGCATC TCATAGTTGCGATTCTTACTGCC chr19: _ PEG3 ENST Exon10-5 CCAAACCTCCAAGAAGTCACAATG CATGTCTGAGCCTTGAATGACAG PEG3 ENST Exon10-6 GCTACCAGCGAAGACCTCAACAC CTTCCCTATGAAGTCTCATATGCTC PEG3 ENST Exon10-7 GAATCGTGCTGCAGAGAGGAATC GCTCTGCAGCCTCTCCATCTGG PEG3 ENST Exon10-8 CACAAGTAGTTCTGAGGATTCAGG CAGTGTGGGTATTCTGGTGTCTG 28

29 Supplementary Table 12 continued. Primers used for PCR amplification and Sanger sequencing Gene Symbol Transcript IDs Coding Exon Number Genomic Region of Interest Forward primer sequence Reverse primer sequence Additional primer sequence for sequencing PEG3 ENST Exon10-9 chr19: _ CCATACTATGACTGCCATGAATGC AGACTGTGGAATCTGCACATTCG PTEN ENST Exon1 chr10: _ CGTTCGGAGGATTATTCGTCTTC CTAGCAACCTGACCAGGGTTAAATG PTEN ENST Exon2 chr10: _ CCTGAATACTGTCCATGTGGAAG * GAAGTCCATTAGGTACGGTAAGCC CCTTTTATTACTCCAGCTATAGTGG (F) PTEN ENST Exon3 chr10: _ CCATAGAAGGGGTATTTGTTGG GCTCTTGGACTTCTTGACTTAATCG PTEN ENST Exon4 chr10: _ GGAAGCACCTGAATTTACAGTACTC * CCTAATTCTTAGTCTATGCTGCAC GCAATACTTTTTCCTAAAACACAACAG (F) PTEN ENST Exon5 chr10: _ CCAGTCCGTATAGCGTAAATTCC GTGAGGTGATGAATATGTTAAGTAG CCACAGTTGCACAATATCCTTTTG (F) CTCAGATCCAGGAAGAGGAAAGG [R] PTEN ENST Exon6 chr10: _ GTTCTTAAATGGCTACGACCCAG CTAGTCAGAATTGGGCTGTATTTGG CCAATGAGTTGAACAACAAAGC [R] PTEN ENST Exon7 chr10: _ CCACTAGAAGTCTAATTTTGGGAC GTCTCACCAATGCCAGAGTAAGC GATCAAGATTGCAGATACAGAATCC (F) PTEN ENST Exon8 chr10: _ GCAAATGTTTAACATAGGTGACAG CAGCAAACAACGAAGAATTAGG CTGCTACGTAAACACTGCTTCG [R] PTEN ENST Exon9 chr10: _ GCTAAGGCACGAGAATTGCTTG GGTCAGGAAAAGAGAATTGTTCC CAACAGAGCAAGACTCTTGTCTC (F) PCDHA13 ENST Exon 1-1 AAATTGGGCCTTGAGAGACAG CTTGCTTTCAGGGAATATGGG PCDHA13 ENST Exon 1-2 GGAGTGTACCATGCTGTCTTCC GATCCAGTCGGTAGGTCAATGC PCDHA13 ENST Exon 1-3 GTGAAGGTGAGGGACATTAACGAC CATATACCACTGCAGGCCATAC PCDHA13 ENST Exon 1-4 TGCAGATATTGGAGTAAACTCGG GGAGTGACAAAGAAGTGATGGTAAC PCDHA13 ENST Exon 1-5 chr5: _ TTAGAAGGCCTGTATGGCCTGC AGCAGACACCGTGAAGATGTG PCDHA13 ENST Exon 1-6 GCCCTAATCAGTGTGTCCGATC CAGCTCGCTCACTGTGCCTC PCDHA13 ENST Exon 1-7 CACATCTTCACGGTGTCTGCTC GTGCTGATCTCGCCAGTGTACAG PCDHA13 ENST Exon 1-8 AGGCACAGTGAGCGAGCTGATG CAGAACCCAGACAAGGAGGAAG PCDHA13 ENST Exon 1-9 CTGGTGGATGTCAATGTTTACTTG GTCCACAGCAGAGTTGTTAAATTCG PCDHA13 ENST Exon 2 chr5: _ CTGTGTGAGGACCCTGATGAAC CTGACCATTTGCGATCTTTCAAG PCDHA13 ENST Exon 3 chr5: _ AGCAGGCAATAAGTAAGTCAGC TCAACTCCTGAAGAGCATTAGG PCDHA13 ENST Exon 4 chr5: _ CTCAGACTTGAACAAACAACTCGTG GCAGATTTCTGAGGTCTCTAAGAGG RADIL ENST Exon2 chr7: _ TGCTCTTGGTGGTCACGATGGTTG GGAAGGCTTCAAACATCCAGGAC RADIL ENST Exon3 chr7: _ GTTTCACTGTGTTGGCCAGGCTG AATGTGGATGACTCCACATGTGC RADIL ENST Exon4-1 TGTCTCCTGGGTCTCTCGTGTG CCTTGAATAGCAGCAGGTAGTAGAG RADIL ENST Exon4-2 chr7: _ GCACATCTCCGTCAACTTCTCC GCTCAGCAGCACAGCACCGTG RADIL ENST Exon4-3 TCTCCGTCAACTTCTCCGAGGTG CAGCTGACACCTTGGGTTCTGG RADIL ENST Exon5 chr7: _ ATAGTCAGCGTCACACAGGATGC ACTCAGAGCTTCCCAGGGTGATG RADIL ENST Exon6 chr7: _ CAGGTTGATGGTTGCAGTTGTTG GCAACTGACACATGGCAGCCTAG RADIL ENST Exon7 chr7: _ TTGAGATGTTGGGTGTCTGGACC TGAAGTGGAGCTTGTCACGGTC RADIL ENST Exon8 chr7: _ ACCTCTGTGCGCCTTCTGAGTTC GGATGCGCTGTGCTGATCTGTC RADIL ENST Exon9 chr7: _ GTCATCGAGCTGTGATGTCCTCAG GGTAGCTTTGAGGTTTCCAGCC 29

30 Supplementary Table 12 continued. Primers used for PCR amplification and Sanger sequencing Gene Symbol Transcript IDs Coding Exon Number Genomic Region of Interest Forward primer sequence Reverse primer sequence Additional primer sequence for sequencing RADIL ENST Exon10 chr7: _ CCAGTAGTGGTTTCTCTGGTTGG * GAGTTTGGAACTTGGGCATGGAC TGAACTGCTGCTGTGACACCGTC (F) RADIL ENST Exon11 chr7: _ AGACACCATCAGAGACCAGTGG ACCTCGGGGCACACTGGCTG GGAATTCAGAAGGGATGAGTCC (F) RADIL ENST Exon12 chr7: _ GGAGTCCTTTGCTGACCTCTG GTCTCAGGTTGCAGGTGTCTGTG RADIL ENST Exon13 chr7: _ CACATTCTGCCTGAACAGCGTC GACAGAGGCAAGAGACAGCAGG RADIL ENST Exon14 and 15 chr7: _ GTCTCTTGCCTCTGTCCTCAAGC GCATGTTATCAACAGGCATGTCC RNF43 ENST Exon2 chr17: _ GACTTGGCTTTCTGAAACGGAAG GAATGCCAATAAGGCAGTATCTACTC RNF43 ENST Exon3 chr17: _ CAGTGGTCACTAGCTTACTAAGAGG CCACTTCTCTCAGACCAGTCATGG RNF43 ENST Exon4 and 5 chr17: _ CTTGCATAGGTAGATGTGAGTGTGC ATGAAGTTGGACTAGAGGGTTTCCAG RNF43 ENST Exon6 chr17: _ CCACTTGCAGGTTCCTCTCCATCC CTGCTGGAAATCAACAAAGACAGACG RNF43 ENST Exon7 chr17: _ GAACTCCTAAGGATGTTCAACTGG CAATAGTGCTCTTTCACTGGAAGG RNF43 ENST Exon8 chr17: _ CTGTTAAGAAAGTTGCCCAACGTC GTGAGGCACTCTGGAGCAGTGTAC RNF43 ENST Exon9-1 GAACAAAGGAGTCCCAGAGCACAG CAAAGTCACTGCTTAGGGAGCTGC RNF43 ENST Exon9-2 AGAGCAGCAGCGCCTGGCAGGAG CTGCTGAGTTGGATCTGGTGACTTGC chr17: _ RNF43 ENST Exon9-3 GTTTCCAGCCATGTCCACTACCAC TCTGGTAGCAGCCTCTTGTCCAGG RNF43 ENST Exon9-4 CAGTACCAGCAGTCTGTTCAACTTG TTGGTTGTCATCTCTGCTGTATCC RNF43 ENST Exon10 chr17: _ CCTTAGCTTTCAATCTAACCACC CACCAGTCCTCTTCCAGTGCTTCTAG ROBO2 ENST Exon1 chr3: _ GATCTTGCGATTTGCTCTTCTTGAC CTACCAACACATTCCACTTACAGCAG ROBO2 ENST Exon2 chr3: _ CAAAGGAGTTGAGATGACTTATGAG TGACCAGACTTCTCTGAAGAAGACC ROBO2 ENST Exon3 chr3: _ GGTGGATGTAGCTATGTTCTCAG GTCTCTTGTAGTCTCATCCAAATG ROBO2 ENST Exon4 chr3: _ GATCAGGAAAGAGAGGACAGCTG ACTGCTTACCATGAGTCATGAGTAAC ROBO2 ENST Exon5 chr3: _ CAGCTGAGGTACAGAGTTAGGTAC * CAAACAGTAAATATGTGGCTTGC CAAACAGTAAATATGTGGCTTGC [R] ROBO2 ENST Exon6 chr3: _ CTAAGTAATTGCACTTTGTGGCTG GATAGAGAGGACACAAAATCTAGG ROBO2 ENST Exon7 chr3: _ CTGTAGTCTCTCTCTGGCACTGAAC GCTATTGCTAGAACATCTGACTCC ROBO2 ENST Exon8 chr3: _ CTTTATCAGTGAAACCCACTGTGTG GTGGTATTCAATCAGGGAGAATTAG ROBO2 ENST Exon9 chr3: _ CAATAAATAGTATGGCCTTAGTATAG CTGTTATGAATGTGTGAGTAACG ROBO2 ENST Exon10 chr3: _ GGTAAGAGTATTATCACAGAGGAAG GTAATGGTGTGTGCAAATTCATGC ROBO2 ENST Exon11 chr3: _ GGAGAAGCAAACATTCACTTACATAC CTCTGTTCTTGAATAAAGTCAGAGAG ROBO2 ENST Exon12 chr3: _ GTGCATGGCACTTTTGTTAGGTG ACACATCTCACCAAGATAAACAGC ROBO2 ENST Exon13 chr3: _ CTTTCATTATGTCCTACCACAGTGG TGAAGTTGTTCACTTTATGGTATGC ROBO2 ENST Exon14 chr3: _ GGTTGGTATCTTAGATACTGTGAGG CATAAGATTTGGTGCAAATGGTTAC ROBO2 ENST Exon15 chr3: _ CTGTCACTGTTGAACACCGTGTG GACCTTGAACTCAGGTTCTAATACC ROBO2 ENST Exon16 chr3: _ CAGAGGTTGGGCTTTAGAAGATG CCATTTGTCAATATGATATGTGTACC ROBO2 ENST Exon17 chr3: _ GGTCAGTGTTCTTAATCACATTTGC CCTTATGGTTATAAAGCGATAGGTC ROBO2 ENST Exon18 chr3: _ CCACTGCTTGTTACTCTCTGTACAATG ATGAAACATCTGGACTTGATTCCTC 30

31 Supplementary Table 12 continued. Primers used for PCR amplification and Sanger sequencing Gene Symbol Transcript IDs Coding Exon Number Genomic Region of Interest Forward primer sequence Reverse primer sequence Additional primer sequence for sequencing ROBO2 ENST Exon19 chr3: _ GTGCCAATGTATCATCAAACCTTG * CTCAAGGTAATGAAGATACAATCG CGTCCACTGTTAATGTATGTATAGC (F) ROBO2 ENST Exon20 chr3: _ CTGTCACTGAGGCTGATAGCTTGG CTCAAGTTACTGGAAACATGCTGG ROBO2 ENST Exon21 chr3: _ CAGCTCTGCTGTTAATGCACTGG GGATTCTCAGTCCTCAATATGTGC ROBO2 ENST Exon22 chr3: _ GATTTACTGTGGTGACACCAACAGC CAGTTACAGTTACAGCTCCAGTTCC ROBO2 ENST Exon23 chr3: _ GTATTCGTAGCCATTGTTTCTGCTG GTGAACTTCTCATGAAAGCCCTAG ROBO2 ENST Exon24 chr3: _ CTACAGAGATGCACCAATATTTGG GCATGGAATGTATTTCTTGAGG ROBO2 ENST Exon25 chr3: _ GGTTCCTATTGTAGGAAATGTTGC CAATGGCCTAAGTTATGACCAAAC ROBO2 ENST Exon26 chr3: _ CACAAACTCATCTATCTGAAGACC * GAAAGAGTTAAGGTGGGTGGTGTA GCACTGTTTAGAGTCATCACTTCC [R] SMAD4 ENST Exon2 chr18: _ CAATTTTCCTTGCAACGTTAGC GCATATTTGTACAGGATGCAC SMAD4 ENST Exon3 chr18: _ GATGTATGACATGGCCAAG CAACTACAATACTCGGTTTTAGC SMAD4 ENST Exon4 chr18: _ GCTACTTCTGAATTGAAATGG CTTGTTAATGTTACTGCCTGC SMAD4 ENST Exon5 chr18: _ GACTTTTGCTGGTAAAGTAGTATGC CCTGTTTACCTATGAAAGATACTAC SMAD4 ENST Exon6 chr18: _ GATAGGCCATGGGTGAGTTACAC CAGTCCTACTTCCAGTCCAGG SMAD4 ENST Exon7 chr18: _ GCAGCAGAATGGATTTACTGG GTTAATGAAACATTAGCAACAGAG SMAD4 ENST Exon8 chr18: _ TAGCACTTGGCAGATAGCAC CATCTGAGAAGTGACCCCAT SMAD4 ENST Exon9 chr18: _ GAAGACATGGAAATTCCTACC GGAGTGCTTACAAATTCTGAG SMAD4 ENST Exon10 chr18: _ GCTCCTGACACATAGTAAGTG CCATTCCTTCCACCCAGATT SMAD4 ENST Exon11 chr18: _ CCAAGCCACCTTTCCTAACTA ATGCAAACAGGGTCATAGGC SMAD4 ENST Exon12 chr18: _ CTTCTTGGCACTTTAGCAGAG CCAGTTTCTGTCTGCTAGGAGC TP53 ENST Exon2 and 3 chr17: _ TGGAAGAGAGAATGTGAAGC GGACTGTAGATGGGTGAAAAG TP53 ENST Exon4 chr17: _ CGTTCTGGTAAGGACAAGG CAAAGGGTGAAGAGGAATC TP53 ENST Exon5 chr17: _ GTAGACGCCAACTCTCTC CTCCACACGCAAATTTCCTTC TP53 ENST Exon6 chr17: _ CATCTACAAGCAGTCACAGC ACCCATTTACTTTGCACATC TP53 ENST Exon7 chr17: _ GCACCTGTAGTCCCAGCTACTCG * TCTGGAGCCTAAGCTCCAGC TGAGAGGTGGATGGGTAGTAG [R] TP53 ENST Exon8 chr17: _ CTTAACCTGTGGCTTCTCC CATCCAGTGGTTTCTTCTTTG TP53 ENST Exon9 chr17: _ CTAAGCGAGGTAAGCAAGC GAGCCATTGTCTTTGAGG TP53 ENST Exon10 chr17: _ TAGGTACTTGAAGTGCAGTTTC CTGGGACCCAATGAGATG TP53 ENST Exon11 chr17: _ GAGGTGCTCAGTAAACATATTTGC CATCTCCCAAACATCCCTCAC 31

32 Supplementary Table 12 continued. Primers used for PCR amplification and Sanger sequencing Gene Symbol Transcript IDs Coding Exon Number Genomic Region of Interest Forward primer sequence Reverse primer sequence Additional primer sequnece for sequencing LAMA2 ENST Exon1 chr6: _ CGAGAGTGGAGGAGTGTCCT CGCTCTCATGGCATTGAACT LAMA2 ENST Exon2 chr6: _ GATGGAAGATTTAGTGGGATC GCCAGGAAGAAGCTCTTAATG LAMA2 ENST Exon3 chr6: _ GAGAATCTGAATTACTCTCCTTC ATCAGCACAGTGGGAGCAAT LAMA2 ENST Exon4 chr6: _ TGAAATGTTGCCAATGAGTT AATGTACTTTTGCTAAATCCTAT LAMA2 ENST Exon5 chr6: _ AACTAATTGGGAGAATGGGA AGCGGCACAGCTTGGATCT LAMA2 ENST Exon6 chr6: _ ACTCTTCCTTTGGGCTAAGA ACTGTGATGGCTGATTTCAA LAMA2 ENST Exon7 chr6: _ ATATACCCACTTCAAGCCTGG CATTCAGTTGAATTGGACTTC LAMA2 ENST Exon8 chr6: _ CAGCCTGGGCGACAGAGT AGTTCAACTGTTTACAGAAGC CTATTATGTATAACAGAAATGA [F] LAMA2 ENST Exon9 chr6: _ ATCTTTACTTTTGTGTAAGCATG GGAGGGAACGCTAATACACT LAMA2 ENST Exon10 chr6: _ CCCTCTACTCTTTGGTTTTAAC TGGATACTTCCAGAAGTCA LAMA2 ENST Exon11 chr6: _ AACACATAGCTTTTGAGCAG ACCTCAAAGGCTTTGAGCAA LAMA2 ENST Exon12 chr6: _ TCTAAATGTTTGTGGTTTGG GGCAATCCAATTCAGTGTGT CAAAAGTGGACACGACCAG[F] LAMA2 ENST Exon13 chr6: _ CCCTATAGAACAATAAAATTCG AGTAGTCCTGTAAGTCAGTGTCA LAMA2 ENST Exon14 chr6: _ ATAGAGCGAGACTCTGTCTCC CTGCATATTTAAAACTGTAAAG GTTCTTATCTGCTCTTTGAA [R] LAMA2 ENST Exon15 chr6: _ GGTGATTGTGAGCACTTTAGAC GCCACAGCTTTTGGTAGATA LAMA2 ENST Exon16 chr6: _ TGCCAATAACACTAAGGTCAG CTTCCTCATATTCATTAGGGAG LAMA2 ENST Exon17 chr6: _ CTCTGCTGTAAAATTGACCA TCTTTGGAATTGGTGTAGAA LAMA2 ENST Exon18 chr6: _ CTCTCCACTGGGCTATATTCAC CTTTATTTCACCTCATTGAGTG LAMA2 ENST Exon19 chr6: _ CACGTTGCGTTTGAGAAAAG GTTCTGGAATATGCAGACTCTG LAMA2 ENST Exon20 chr6: _ CCTAACAGGGCACTTAACAA GTTTAAGGTCAAATGGTAATGT LAMA2 ENST Exon21 chr6: _ TCAGTGAGGAATCCTGATAACT TACGTTGTATCAATCTGTGCTT LAMA2 ENST Exon22 chr6: _ TATTGAACAGCTCCTTTCTGA ACTTCATCATCTTTCCTACAAGT LAMA2 ENST Exon23 chr6: _ CCCAAGTGACTAAACTAGAATAGTAC CTCCATGTCTGGCAATGAGA LAMA2 ENST Exon24 chr6: _ GAGTATGCTCCCGTTATGCATT CTGTTCCTAAATGTTCACAGAGG LAMA2 ENST Exon25 chr6: _ GTGAGTTCTGTTGCTTGTGAG GATTGCATACTTGAGTTTGCC LAMA2 ENST Exon26 chr6: _ AGAGAAGATCTCCATTTGGAAC GTATATCCAAGAAGTCTTCTCGG LAMA2 ENST Exon27 chr6: _ CATTCGAGGTGGGACACC GGGAAGCCTCCATGGTTTA LAMA2 ENST Exon28 chr6: _ CAATAGAACATACTTGAGGTTGAC CTGTGTCTCACACTGTGAGTG LAMA2 ENST Exon29 chr6: _ CAGCCACTGAAGAGCTCAAG AATGCAGAATCTGTGAATATCTT LAMA2 ENST Exon30 chr6: _ AGACTGAGTTCTCTGTCAGTGAT GAACCATAGAGCATTATATACTCAC LAMA2 ENST Exon31 chr6: _ TTCATTGTGACGTCCTAGCC GACAAATTCTAAGATACACAAGTGG LAMA2 ENST Exon32 chr6: _ TCACGGCATAATCTTGATTT GGGAGAGTCACTCTTTCTCAT 32

33 Supplementary Table 12 continued. Primers used for PCR amplification and Sanger sequencing Gene Symbol Transcript IDs Coding Exon Number Genomic Region of Interest Forward primer sequence Reverse primer sequence Additional primer sequnece for sequencing LAMA2 ENST Exon33 chr6: _ CCTCAGTTTATATTATCGCCTCT AACAGTTGTGTCCCTTGATTTG LAMA2 ENST Exon34 chr6: _ CTTTGAAGGTGAGAAGGCTAAG GAAGCAGAATTAGCAAGTCAGA LAMA2 ENST Exon35 chr6: _ TTAATTGTATACGTGAGCATGG TCTCAGCAGATAATACCCTTG LAMA2 ENST Exon36 chr6: _ TTCCCAGCAGGAACACTCA AGCAAATATCTCCAGGTTGG LAMA2 ENST Exon37 chr6: _ GGTCTCAGGTACCAGTTTTCA CTTTCAGAGTTGTCACTCTTGAC LAMA2 ENST Exon38 chr6: _ CATCTAGCCACATTTCAACATG ACACTCATGTCCATTGGGAG LAMA2 ENST Exon39 chr6: _ GACAGAGCAAGACTCTGTCTCA TGTAGCCTAGGGCTATGTTCA GATGCTGCTGTAGCAAAGC [R] LAMA2 ENST Exon40 chr6: _ GGAATTGTCATAGATATATTGGC AGTGGTAGTGGGTGACTGAGA LAMA2 ENST Exon41 chr6: _ CAGTCTCCTAACTTTAACTACCGA ACTTGAAGTGAGATAGGTAGACAAC LAMA2 ENST Exon42 chr6: _ GTTCAACATGTAGAAGCATCCA AATACCATAATGCAAAGTATGGT LAMA2 ENST Exon43 chr6: _ TACACCTGAATGAGACAAAGTTC AGAAGACTTGGATTCCAGCT LAMA2 ENST Exon44 chr6: _ GGCCACTAAATTATATATTTTGTG CCACTTCGCCTGACCAAAC CTGTAACAGTGGAAAGAATAGCT[R] LAMA2 ENST Exon45 chr6: _ TTGACATTTGCCATCACACA TCCTACCCCAAATCGCTTC LAMA2 ENST Exon46-1 ACATGGAACTGTGAACAGCTC TGTAAGGTAAGACGTTGAGGTT chr6: _ LAMA2 ENST Exon46-2 GAGTATAATGGTGTCTGCATATGG ATCTGAAAGCCTACGTTAGACC LAMA2 ENST Exon47 chr6: _ GAAGCTCCTAATCATTTCACATGTC CCTCTGGTCAATACCTGGAAGG LAMA2 ENST Exon48 chr6: _ GATCAGACTCTGAGTATTGATTCG GTGTTTCCAGGTTGAATCGTC LAMA2 ENST Exon49 chr6: _ GTAGTAGCTCTAAGGATACGGACAG CAGGCAGGGACTTGAAGATTC LAMA2 ENST Exon50 chr6: _ GTAGAGATGGAGATAAGTGGAAGG CTGCCAGACAATCATCCCAG LAMA2 ENST Exon51 chr6: _ GCAATTTAGCCACAAGACCCTAAC CCCAAATCACCTATAATCTAAGCC LAMA2 ENST Exon52 chr6: _ GCTAAATATAGTGCAAGTGCTTGAG CCTGGTGTTGTTCTAACATGCAAC LAMA2 ENST Exon53 chr6: _ CCTTTGTAGATCCCTGTGAAATG CTTGCTCGTGAAACATACTATGAAC LAMA2 ENST Exon54 chr6: _ CCAGTAGACAATGGAAACCAGAGT ATGTGCTATAAAGAGCTATACATTTGG LAMA2 ENST Exon55 chr6: _ TCTTCAACTGCTCTTAAACTTTCC GGAAATATGGAATGTTCAGTCC LAMA2 ENST Exon56 chr6: _ CACTTTCCAGATCATCTCACAAGC ATGTTAAGGGTAGAACTCCTAAGG LAMA2 ENST Exon57 chr6: _ CTTAGTGTAGAATTCCTAAGCATCC GCATAGCTGTAGACAATGTAGATGC LAMA2 ENST Exon58 chr6: _ GCATCTACATTGTCTACAGCTATGC GGTAAGTCATTCAATGAAGATGTAAG LAMA2 ENST Exon59 chr6: _ GATTCACACGCACTGAGCACTC GACTTTATCAGGCTACTGATTTCC LAMA2 ENST Exon60 chr6: _ CATCTCTGAGAAGGTTTTACTCTCC GCAAACCTACACAGGTCTGAGCAC LAMA2 ENST Exon61 chr6: _ GCATACTAAGCACTGTGTACATTG CGACAGAGCGAGACTCTGTCTC LAMA2 ENST Exon62 chr6: _ CAAGTTGAGAAGCACAACACAGG AAGGATACTAGTTTAGGACTCTAGGAAC LAMA2 ENST Exon63 chr6: _ CATGATGCAGTGGTGGAAGATAG CTCCCAACAAAGAATACAGCCG LAMA2 ENST Exon64 chr6: _ GATTCTGGCTGATGTCTTTCC GGAAGCAAGTACACATAAGCACAC LAMA2 ENST Exon65 chr6: _ CAGCATTCCAATTTAATCTCAAGC GCACCATCTGAATACAGAAAGAATTC 33

34 Supplementary Table 12 continued. Primers used for PCR amplification and Sanger sequencing Gene Symbol Transcript IDs Coding Exon Number Genomic Region of Interest Forward primer sequence Reverse primer sequence Additional primer sequnece for sequencing XIRP2 ENST Exon2 chr2: _ CCAAGAGCTGGTGCTCCTAGAG CAGGACATCTGTCTAGAATACTGG XIRP2 ENST Exon3 chr2: _ CTCTTTCATTTCTGCCTTCTAACC GTCAAGTGCATTGTTTCAAACCTC XIRP2 ENST Exon4 chr2: _ GCTACAGTCCATTTTACGAGATC CACACACTATTTTTGTGCTCTTTC XIRP2 ENST Exon5 chr2: _ GACATACTGAAATAATGGTGTATGTG GCTACAGTGTTTGTTAATGAGTAATG XIRP2 ENST Exon6 chr2: _ CAGAAATTGTCACTGAAATTGCAC AGAATTCCAACTTGAAAGTGGAG XIRP2 ENST Exon7 chr2: _ GCATCTCTCAATGTACACTAAAGTC GACTGTGCTTTGGAAGTCATCC XIRP2 ENST Exon8 chr2: _ GTATTGGGATTACAGGAATGAGC GTTGAAAAGCCACCTCAAGTATTC XIRP2 ENST Exon9-1 TCACATATTGTTCCCAATGGAG CCAAGTATTCTCTTTCTGAGTTCAG XIRP2 ENST Exon9-2 CCCAAAGATGTATATTCCAAGC GAATGAAGCTGTCCAAGTTGATC XIRP2 ENST Exon9-3 CTTCTGACACTGTAGAAAATGCAG TCAATGATGACCTCTTCTGACTC XIRP2 ENST Exon9-4 GATGTAAGAACAGCACGGTGG CTCTTGTAACTCCTTCCACCTC XIRP2 ENST Exon9-5 ACAGTCCTGATATAGGAAAGCTTC CCATTTACAGGTTTTGACATCC XIRP2 ENST Exon9-6 GACCAGTTTGATGAAAGCATTC CATTGCCTTTCTGAATATCTTCAG XIRP2 ENST Exon9-7 ACAGGAGGAGATCCAAGGTGG CTGAATGTCTTCTTGTGTGACAG XIRP2 ENST Exon9-8 AGAAGAATCTGACTATATCAGCACC CTTCCATAATAGAATCGAAAGTTCG XIRP2 ENST Exon9-9 GACAAGTAGACAATTCTTTGAGTCTG ACATTTCCCTTCTCTTCTTCAC XIRP2 ENST Exon9-10 AAGAAACCTTAGAAGATCTCTACTCTC TCATTTGACTCTGTGTGGAGTTG XIRP2 ENST Exon9-11 GAAAATGATGGTGACACAATTGAGC CAATATCACCAGGGATGGCATC chr2: _ XIRP2 ENST Exon9-12 TCAGAGTACATTTGGTAAGATACCC TCACGATCTATGACAATCTTTACAG XIRP2 ENST Exon9-13 CATCAGGTTGCTGTCCAGAGG GCTTGGAGCTTTGTGTATCAG XIRP2 ENST Exon9-14 GAATCAGACAGGGCAGTGAGAG CATTAGAAGGTGGTGGAGGAGG XIRP2 ENST Exon9-15 GCAAGACACATCCAATGTAACAG GTTTGGGTTTGGGCAATGTG XIRP2 ENST Exon9-16 CCAGTAGATGAGAAATCTGAAAGAGA GGCTTTGAGTTTTAACAATGTTGTAG XIRP2 ENST Exon9-17 CTGCAAACTGTCTCTCACACACA ATGACTTTCATAGCTGTGGTCTG XIRP2 ENST Exon9-18 CATTATGCAATCCAAATCAGC GTATGCCCTTGACTTCGAGAC XIRP2 ENST Exon9-19 GGACCATCAATGATTGGTCG GCTGACATGAACATTAGATACTTTGG AGTCCCTACCACGAGTTTCTATC XIRP2 ENST Exon9-20 CAGTTCACATTGCCATGGAG AGTCCCTACCACGAGTTTCTATC XIRP2 ENST Exon9-21 GAAATTCACAGAGCAAACACTTCC CTCGCAGTTCTCTAGGATGTTTGC XIRP2 ENST Exon9-22 ACCGAACTGTTCAAATGGCTG GTTTGGTTGTTTCCACGCACTC XIRP2 ENST Exon10 chr2: _ GAGCTTTTCTACTTTAACCGCATGG GCTATACATAGAGTGCAGCTCCTG *Primer sequence used for PCR amplification only, but not for sequencing as regions of interest present high GC contents or nucleotide repeats. (F) or [R] Additional primers used for sequencing to overcome primer slippage and to obtain high coverage in region of interest.. 34

35 Supplementary Figure 1. Distribution of mutations in 8 O. viverrini-associated CCA exomes. Pie chart illustrates the types of mutations present in CCA exomes and the number of each mutation type is reflected in the table. 35

36 Code SMAD4 TP53 KRAS GNAS RNF43 ROBO2 PEG3 MLL3 CDKN2A PTEN NCD80 RADIL PCDHA13 LAMA2 XIRP2 W A U A B B Y Y Y T R A142 + W Y035 + Y002 + R134 + A162 + A107 + A042 + A119 + A120 + B032 + B083 + Y072 + R B A035 + U027 + Y019 + T151 + B T157 + Y W Y057 + Y023 + R100 + A106 + T003 + A T160 + A039 B087 Y140 Y123 Y074 Y065 Y033 Y032 A043 A028 A128 B048 B113 Supplementary Figure 2: Summary of commonly mutated genes in 54 O. viverrini-associated CCA samples. Left column indicates the patient identity and top row are genes validated in this study. Red boxes indicate genes mutated in the respective samples. Region boxed with black border indicate samples with mutated genes excluding TP53, KRAS and SMAD4. 36

37 Supplementary Figure 3: Kaplan-Meier plots of overall survival of patients with tumors containing KRAS and RNF43 mutations. (a) Five patients with RNF43 gene mutations vs. 48 patients with wildtype (P < 0.001). (b) Nine patients with KRAS gene mutation vs. 44 patients with wild-type (P = 0.014). NB: Peri-operative death cases (survival time < 30 days) were excluded from analysis. 37

38 38