The American Journal of Human Genetics Supplemental Data Genomic Analyses Reveal Mutational Signatures and Frequently Altered Genes in Esophageal Squa

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1 The American Journal of Human Genetics Supplemental Data Genomic Analyses Reveal Mutational Signatures and Frequently Altered Genes in Esophageal Squamous Cell Carcinoma Ling Zhang, Yong Zhou, Caixia Cheng, Heyang Cui, Le Cheng, Pengzhou Kong, Jiaqian Wang, Yin Li, Wenliang Chen, Bin Song, Fang Wang, Zhiwu Jia, Lin Li, Yaoping Li, Bin Yang, Jing Liu, Ruyi Shi, Yanghui Bi, Yanyan Zhang, Juan Wang, Zhenxiang Zhao, Xiaoling Hu, Jie Yang, Hongyi Li, Zhibo Gao, Gang Chen, Xuanlin Huang, Xukui Yang, Shengqing Wan, Chao Chen, Bin Li, Yongkai Tan, Longyun Chen, Minghui He, Sha Xie, Xiangchun Li, Xuehan Zhuang, Mengyao Wang, Zhi Xia, Longhai Luo, Jie Ma, Bing Dong, Jiuzhou Zhao, Yongmei Song, Yunwei Ou, Enming Li, Liyan Xu, Jinfen Wang, Yanfeng Xi, Guodong Li, Enwei Xu, Jianfang Liang, Xiaofeng Yang, Jiansheng Guo, Xing Chen, Yanbo Zhang, Qingshan Li, Lixin Liu, Yingrui Li, Xiuqing Zhang, Huanming Yang, Dongxin Lin, Xiaolong Cheng, Yongjun Guo, Jun Wang, Qimin Zhan, and Yongping Cui

2 Figure S1. Fold coverage of whole genome and exome in the sequenced normal and tumor samples in 104 ESCC cohort. (A) the box plot depicts the distribution of mean coverage in whole-genome sequencing set. (B) the box plot depicts the distribution of fraction of bases covered by at least 1 reads, 4 reads, 10 reads and 20 reads across the sequencing samples in whole-genome sequencing set. N, normal samples; T, tumor samples. (C) the box plot depicts

3 the distribution of mean coverage in whole-exome sequencing set. (D) the box plot depicts the distribution of fraction of bases covered by at least 1 reads, 4 reads, 10 reads and 20 reads across the sequencing samples in whole-exome sequencing set. All samples were calculated with an average estimated tumor content of 40-50%. (E) Sequencing and analytical pipeline for determining somatic mutations in this study.

4 Figure S2. Mutation spectrum in 104 ESCC patients. (A) Detailed spectrum of coding-region mutation subtypes in ESCC. Base substitutions are divided into six categories represented by a different color. The entire set (n=104) shows a predominance of spontaneous deamination in C>T (red). (B) Comparison of mutation spectrum between EAC and ESCC (Student s t-test). Mutations were from coding regions of 16 EAC and 14 ESCC determined by WGS. Left box represents EAC and right one represents ESCC for each color.

5 Figure S3. Identifying the number of process operative in 192 ESCC genomes (104 from this study and 88 from ref.6) based on reproducibility of signatures and low error for reconstructing the original catalogs.

Copy number amplification (red) and depletion (blue) are plotted as a function

6 Figure S4. Identified copy number alterations in WGS set. (A) Copy-number profiles in whole-genome sequencing set of 14 ESCCs. Copy number amplification (red) and depletion (blue) are plotted as a function distance along the normal genome. (B) One significant, focally amplified (red) region viewed by IGV is plotted along the chromosome.

7 Figure S5. Amplifications of SOX2 gene identified by IGV. Focal amplification in 3T06, marked with red star, only contains SOX2 gene.

8 Figure S6. Amplified genes identified using GISTIC in ESCC. Knockdown of CBX4 in KYSE2 and KYSE510 cells significantly inhibited cell proliferation, as measured by MTT assay (A), colony formation assays (B), and cell invasion (C). Knockdown of CBX4 is demonstrated by western blotting (bottom of a); β-actin was used as a loading control.

12 Figure S7. Validation of mutations via Sanger sequencing. (A) Sanger sequencing of tumors harboring AJUBA somatic mutations. Alignment of AJUBA mutation (152delG) from IGV (Intergative Genomics Viewer) are shown. (B) Sanger sequencing of tumors harboring ZNF750 somatic mutations. Alignment of ZNF750 mutation (S503C) from IGV (Intergative Genomics Viewer) are shown. S503C was identified in 3N52-VS-3T52 patient whose tumor genomic DNA was unavailable for Sanger sequencing validation. (C) Sanger sequencing of tumors harboring PTCH1 somatic mutations.

13 Figure S8. The effect on cell proliferation (MTT assay, A), colony formation (B), migration (C), and invasion (D) of AJUBA knockdown in KYSE140 and KYSE510 cells. These cell lines harbor higher level of endogenous AJUBA expression. Data represent the mean ± SD; three independent experiments were done; each experiment was performed in triplicate. Statistical analysis was done using a two-sided t-test. **P<0.01, *P<0.05. WT or mutant AJUBA expression (bottom of A) was confirmed by western blotting using an anti-ajuba antibody recognizing the N-terminus of the AJUBA protein.

Figure S9. ZNF750 copy number deletion and its function. (A) Copy number loss in ZNF750 locus viewed by IGV. (B) Focal deletions at the ZNF750 locus (17q25.3) in three tumors are shown from IGV.

14 Figure S9. ZNF750 copy number deletion and its function. (A) Copy number loss in ZNF750 locus viewed by IGV. (B) Focal deletions at the ZNF750 locus (17q25.3) in three tumors are shown from IGV. Copy number was shown in Y axis. (C) ZNF750 copy number was assayed by qpcr in seven tumors (blue) identified harboring deletions in 14 WGS or 90 WES set. The RNase P gene was used as reference normal (red). Data are mean ± SD. Red asterisks indicate tumors also harbor ZNF750 mutations. Three primers were used to for validation of ZNF750 copy number deletion and all assays were performed in triplicate. (D, E) Effects on cell proliferation (MTT assay, D) and cell migration (E) of stable knockdown of endogenous ZNF750 followed by over expression of ZNF750 wild-type or ZNF750-S70X (p.ser70*), ZNF750-W207X (p.trp207*) mutants in KYSE150 (left) and KYSE140 (right) cells. ZNF750 knockdown was demonstrated by western blotting using an anti-znf750 antibody recognizing the ZNF750 C-terminus (left blot below D). Expression of ZNF750 mutants was confirmed using an anti-flag antibody (right blot below D). β-actin was used as the loading control. Data represent the mean ± SD; three

15 independent experiments were done; each experiment was performed in triplicate. Statistical analysis was done using a two-sided t-test. **P<0.01, *P<0.05.

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