Supplementary Table S1. Summary of the Six Next-Generation Sequencing (NGS) Studies Containing 241 Paired Melanoma Tumor/Normal Samples

Supplementary Table S1. Summary of the six next-generation sequencing (NGS) studies containing 241 paired melanoma tumor/normal samples

Study NGS # paired tumor-normal Tumor subtype Reference ID technology samples

1 Whole genome 25 23 cutaneous, 2 acral Berger et al., 2012 (1)

95 cutaneous, 5 acral, 2 mucosal, 1 uveal, and 18 2 Whole exome 121 Hodis et al., 2012 (2) unknown

61 cutaneous, 14 acral, 7 mucosal, 5 uveal, and 12 Krauthammer et al., 2012 3 Whole exome 99* unknown (3)

4 Whole exome 7 7 cutaneous Nikolaev et al., 2012 (4)

5 Whole exome 8 8 cutaneous Stark et al., 2012 (5)

6 Whole exome 14 14 cutaneous Wei et al., 2011 (6)

187 cutaneous, 19 acral, 9 mucosal, 6 uveal, and 30 Total 241# unknown

*48 tumor samples without normal samples were excluded from our study. #23 paired samples in Berger et al. (2012) were used in Hodis et al. (2012). In addition, there were 10 samples without any mutations in Krauthammer et al. (2012). These samples were excluded in our analysis.

Supplementary Table S2. Summary of known driver mutations detected in the 241 melanoma samples

Mutation* Type # samples BRAF GNAQ GNA11 KIT NRAS present

Acral 17 3 1 0 0 0 2

Mucosal 7 2 1 0 0 0 1

Uveal 6 3 0 0 3 0 0

Cutaneous 182 138 99 0 0 1 38

Unknown 29 26 20 0 0 0 6

Total 172 121 241 0 (0%) 3 (1.2%) 1 (0.4%) 47 (19.5%) (frequency) (71.3%) (50.2%)

*Includes the somatic point mutations identified by the Vanderbilt melanoma SNaPshot assay and known to be functional and actionable (7).

Supplementary Table S3. Mutated genes significantly associated with BRAF-mutated samples (frequency≥ 10% and p-value < 0.05) in the Hodis et al. study

# samples with BRAF # samples without mutation BRAF mutation Frequency Frequency p- Gene (N = 76) (%) (N = 45) (%) value

PIK3C2G 9 11.8 0 0% 0.013

Supplementary Table S4. Mutated genes significantly associated with NRAS-mutated samples (frequency≥ 10% and p-value < 0.05) in the Hodis et al. study

# samples with NRAS # samples without mutation NRAS mutation Frequency Frequency p- Gene (N = 31) (%) (N = 90) (%) value

PIK3R4 5 16.1 1 1.1 0.004

PI4KA 6 19.4 4 4.4 0.018

PPP6C 6 19.4 5 5.6 0.031

TRPM6 10 32.3 13 14.4 0.031

KALRN 8 25.8 9 10.0 0.034

TSC2 4 12.9 2 2.2 0.037

Genes were ordered by p-values

Supplementary Table S5. Mutated genes significantly associated with BRAF-mutated samples (frequency≥ 10% and p-value < 0.05) in the Krauthammer et al. study

# samples with BRAF # samples without BRAF mutation mutation Frequency Frequency p- Gene (N = 34) (%) (N = 55) (%) value

STK33 4 11.8 0 0 0.019

NF1 7 20.6 3 5.5 0.034

Genes were ordered by p-values

Supplementary Table S6. Mutated genes significantly associated with NRAS-mutated samples (frequency≥ 10% and p-value < 0.05) in the Krauthammer et al. study

# samples with NRAS # samples without mutation NRAS mutation Frequency Frequency p- Gene (N = 19) (%) (N = 70) (%) value

TTN 10 52.6 19 27.1 0.036

ALPK1 2 10.5 0 0 0.044

MAPK14 2 10.5 0 0 0.044

Genes were ordered by p-values.

Supplementary Table S7. Mutated genes significantly associated with BRAF-mutated samples identified by the meta-analysis but not significantly BRAF-associated in the Hodis et al. study (corresponds with Table 1)

# samples with BRAF # samples without BRAF mutation mutation Frequency Frequency p- Gene (N = 76) (%) (N = 45) (%) value

TTN 63 82.9 34 75.6 0.227

TP53 17 22.4 5 11.1 0.093

COL1A1 10 13.2 5 11.1 0.490

Supplementary Table S8. Mutated genes significantly associated with NRAS-mutated samples identified by the meta-analysis but some of them not significantly NRAS-associated in the Hodis et al. study (corresponds with Table 2)

Gene # samples with NRAS Frequency # samples without NRAS Frequency p- mutation (%) mutation (%) value

(N = 31) (N = 90)

TTN 27 87.1 70 77.8 0.197

PPP6C 6 19.4 5 5.6 0.031

KALRN 8 25.8 9 10.0 0.034

PIK3R4 5 16.1 1 1.1 0.004

TRPM6 10 32.3 13 14.4 0.031

GUCY2C 4 12.9 5 5.6 0.170

PRKAA2 5 16.1 6 6.7 0.114

Supplementary Table S9. Mutated genes significantly associated with BRAF-mutated samples as identified by the meta-analysis but not significantly BRAF-associated in the Krauthammer et al. study (corresponds with Table 1)

# samples with BRAF # samples without BRAF mutation mutation Frequency Frequency p- Gene (N = 34) (%) (N = 55) (%) value

TTN 12 35.3 17 30.9 0.420

TP53 6 17.6 5 9.1 0.194

COL1A1 2 5.9 1 1.8 0.324

Supplementary Table S10. Mutated genes significantly associated with NRAS-mutated samples as identified by the meta-analysis but most of them not significantly NRAS- associated in the Krauthammer et al. study (corresponds with Table 2)

Gene # samples with NRAS Frequency # samples without NRAS Frequency p- mutation (%) mutation (%) value

(N = 19) (N = 70)

TTN 10 52.6 19 27.1 0.036

PPP6C 3 15.8 5 7.1 0.226

KALRN 5 26.3 11 15.7 0.227

PIK3R4 1 5.3 2 2.9 0.518

TRPM6 3 15.8 10 14.3 0.559

GUCY2C 2 10.5 2 2.9 0.199

PRKAA2 2 10.5 3 4.3 0.289

Supplementary Table S11. Meta-analysis defined mutated genes associated with BRAF- mutated samples (frequency≥ 10%, p-value < 0.05 is shown in red) by histological subtype

# samples with # samples without Type BRAF mutation (N BRAF mutation (N = Gene = Total number) Frequency Total number) Frequency p-value

TTN Total 84 (N = 130) 64.6% 54 (N = 111) 48.7% 0.009

Acral 0 (N = 1) 0.0% 0 (N = 16) 0.0% 1.000

Mucosal 1 (N = 1) 100% 0 (N = 6) 0.0% 0.143

Uveal 0 (N = 0) 0.0% 1 (N = 6) 16.7% 1.000

Cutaneous 69 (N = 108) 63.9% 48 (N = 74) 64.9% 0.614

Unknown 14 (N = 20) 70.0% 5 (N = 9) 55.6% 0.364

TP53 Total 28 (N = 130) 21.5% 11 (N = 111) 9.9% 0.011

Acral 0 (N = 1) 0.0% 0 (N = 16) 0.0% 1.000

Mucosal 0 (N = 1) 0.0% 0 (N = 6) 0.0% 1.000

Uveal 0 (N = 0) 0.0% 0 (N = 6) 0.0% 1.000

Cutaneous 22 (N = 108) 20.4% 11 (N = 74) 14.9% 0.228

Unknown 6 (N = 20) 30.0% 0 (N = 9) 0.0% 0.082

COL1A1 Total 17 (N = 130) 13.1% 6 (N = 111) 5.4% 0.034

Acral 0 (N = 1) 0.0% 0 (N = 16) 0.0% 1.000

Mucosal 0 (N = 1) 0.0% 0 (N = 6) 0.0% 1.000

Uveal 0 (N = 0) 0.0% 0 (N = 6) 0.0% 1.000

Cutaneous 14 (N = 108) 13.0% 6 (N = 74) 8.1% 0.217

Unknown 3 (N = 20) 15.0% 0 (N = 9) 0.0% 0.312

Genes were ordered by p-values.

Supplementary Table S12. Meta-analysis defined mutated genes associated with NRAS- mutated samples (frequency≥ 10%, p-value < 0.05 is shown in red) by histological subtype

Gene Type # samples with Frequency # samples without Frequency p-value NRAS NRAS mutation mutation (N = (N = Total Total number) number)

TTN Total 37 (N = 51) 72.6% 101 (N = 190) 53.2% 0.009

Acral 0 (N = 2) 0.0% 0 (N = 15) 0.0% 1.000

Mucosal 0 (N = 1) 0.0% 1 (N = 6) 16.7% 1.000

Uveal 1 (N = 1) 100% 0 (N = 5) 0.0% 0.167

Cutaneous 32 (N = 41) 78.0% 85 (N = 141) 60.3% 0.026

Unknown 4 (N = 6) 66.7% 15 (N =23) 65.2% 0.669

PPP6C Total 9 (N = 51) 17.7% 11 (N = 190) 5.8% 0.011

Acral 0 (N = 2) 0.0% 0 (N = 15) 0.0% 1.000

Mucosal 0 (N = 1) 0.0% 1 (N = 6) 16.7% 1.000

Uveal 0 (N = 1) 0.0% 0 (N = 5) 0.0% 1.000

Cutaneous 8 (N = 41) 19.5% 9 (N = 141) 6.4% 0.017

Unknown 1 (N = 6) 16.7% 1 (N =23) 4.3% 0.002

KALRN Total 14 (N = 51) 27.5% 24 (N = 190) 12.6% 0.012

Acral 0 (N = 2) 0.0% 1 (N = 15) 6.7% 1.000

Mucosal 0 (N = 1) 0.0% 0 (N = 6) 0.0% 1.000

Uveal 0 (N = 1) 0.0% 0 (N = 5) 0.0% 1.000

Cutaneous 12 (N = 41) 29.3% 21 (N = 141) 14.9% 0.034

Unknown 2 (N = 6) 33.3% 2 (N =23) 8.7% 0.180

PIK3R4 Total 6 (N = 51) 11.8% 5 (N = 190) 2.6% 0.013

Acral 0 (N = 2) 0.0% 0 (N = 15) 0.0% 1.000

Mucosal 0 (N = 1) 0.0% 0 (N = 6) 0.0% 1.000

Uveal 0 (N = 1) 0.0% 0 (N = 5) 0.0% 1.000

Cutaneous 5 (N = 41) 12.2% 5 (N = 141) 3.5% 0.048

Unknown 1 (N = 6) 16.7% 0 (N =23) 0.0% 0.207

TRPM6 Total 14 (N = 51) 27.5% 26 (N = 190) 13.7% 0.020

Acral 0 (N = 2) 0.0% 0 (N = 15) 0.0% 1.000

Mucosal 0 (N = 1) 0.0% 0 (N = 6) 0.0% 1.000

Uveal 0 (N = 1) 0.0% 0 (N = 5) 0.0% 1.000

Cutaneous 13 (N = 41) 31.7% 23 (N = 141) 16.3% 0.028

Unknown 1 (N = 6) 16.7% 3 (N =23) 13.0% 0.627

GUCY2C Total 7 (N = 51) 13.7% 8 (N = 190) 4.2% 0.021

Acral 0 (N = 2) 0.0% 0 (N = 15) 0.0% 1.000

Mucosal 0 (N = 1) 0.0% 0 (N = 6) 0.0% 1.000

Uveal 0 (N = 1) 0.0% 0 (N = 5) 0.0% 1.000

Cutaneous 6 (N = 41) 14.6% 7 (N = 141) 5.0% 0.045

Unknown 1 (N = 6) 16.7% 1 (N =23) 4.3% 0.377

PRKAA2 Total 7 (N = 51) 13.7% 10 (N = 190) 5.3% 0.043

Acral 0 (N = 2) 0.0% 0 (N = 15) 0.0% 1.000

Mucosal 0 (N = 1) 0.0% 0 (N = 6) 0.0% 1.000

Uveal 0 (N = 1) 0.0% 0 (N = 5) 0.0% 1.000

Cutaneous 6 (N = 41) 14.6% 10 (N = 141) 7.1% 0.120

Unknown 1 (N = 6) 16.7% 0 (N =23) 0.0% 0.207

Genes were ordered by p-values.

Supplementary Table S13. Meta-analysis identified guanine nucleotide-binding protein α- subunit (GNA) mutations in the 69 pan-negative melanoma samples

Amino acid Tumor Gene cDNA change Tumor name Reference change subtype

GNA12 c.1129G>A p.D377N MEL-Ma-Mel-48-Tumor Cutaneous Hodis et al. (2)

Krauthammer et GNA15 c.653A>C p.K218T YUDAB Cutaneous al. (3)

GNA15 c.517G>A p.E173K 96T Cutaneous Wei et al. (6)

GNA15 c.1096C>T p.R366C 18T Cutaneous Wei et al. (6)

MEL-JWCI-WGS-34- GNAI2 c.995A>T p.N332I Cutaneous Hodis et al. (2) Tumor

GNAI2 c.989C>T p.T330I MEL-JWCI-WGS-7-Tumor Cutaneous Hodis et al. (2)

GNAI2 c.488T>A p.I163N MEL-Ma-Mel-103b-Tumor Cutaneous Hodis et al. (2)

GNAI2 c.491C>T p.A164V MEL-Ma-Mel-103b-Tumor Cutaneous Hodis et al. (2)

Krauthammer et GNAI2 c.142A>T p.T48S YURTHE Unknown al. (3)

Krauthammer et GNAI2 c.536G>A p.R179H YUHOIN Mucosal al. (3)

Krauthammer et GNAO1 c.724A>C p.N242H YUKLAB Cutaneous al. (3)

MEL-JWCI-WGS-34- GNAS c.229C>T p.P77S Cutaneous Hodis et al. (2) Tumor

Krauthammer et GNAT1 c.586G>A p.D196N YUBER Cutaneous al. (3)

GNAT2 c.722A>C p.N241T MEL-JWCI-WGS-7-Tumor Cutaneous Hodis et al. (2)

Krauthammer et GNAT2 c.482G>A p.R161Q YUPROST Cutaneous al. (3)

Krauthammer et GNAT2 c.25G>A p.D9N YUDAB Cutaneous al. (3)

GNAT3 c.201G>A p.M67I MEL-JWCI-WGS-7-Tumor Cutaneous Hodis et al. (2)

GNAT3 c.400C>T p.P134S MEL-Ma-Mel-114-Tumor Cutaneous Hodis et al. (2)

GNAZ c.451G>A p.D151N 18T Cutaneous Wei et al. (6)

Supplementary Table S14. Meta-analysis identified mutations in 12 potential cancer genes in pan-negative melanoma samples

This table is an EXCEL file (Supplementary Table S14.xlxs)

Supplementary Table S15. Summary of Meta-analysis identified genes with mutations enriched in pan-negative sun-exposed melanoma samples (mutation frequency ≥ 10% and p-value < 0.05)

# pan-negative sun- exposed # driver mutation- melanomas (N = Frequency positive sun-exposed Frequency Gene 44) (%) melanomas (N = 138) (%) p-value

STK31 18 40.9 13 9.4 7.55E-06

ALK 12 27.3 4 2.9 9.35E-06

DGKI 11 25.0 8 5.8 0.001

KDR 10 22.7 7 5.1 0.001

MAPK1 5 11.4 0 0.0 0.001

MARK1 6 13.6 1 0.7 0.001

PAK7 14 31.8 18 13.0 0.001

ADAMTS18 16 36.4 19 13.8 0.002

EPHA7 11 25.0 9 6.5 0.002

ERBB4 16 36.4 19 13.8 0.002

EPHA2 5 11.4 1 0.7 0.003

NF1 12 27.3 12 8.7 0.003

PIP5K1B 6 13.6 2 1.4 0.003

TYK2 6 13.6 2 1.4 0.003

EPHA4 7 15.9 4 2.9 0.005

RAC1 7 15.9 4 2.9 0.005

TAF1L 11 25.0 11 8.0 0.005

MYLK 11 25.0 12 8.7 0.007

EPHB2 7 15.9 5 3.6 0.009

CDC42BPA 5 11.4 2 1.4 0.010

DGKH 5 11.4 2 1.4 0.010

MAP3K11 5 11.4 2 1.4 0.010

PIK3R5 5 11.4 2 1.4 0.010

TSC1 5 11.4 2 1.4 0.010

GRIN2A 18 40.9 30 21.7 0.012

DCLK1 6 13.6 4 2.9 0.014

SYK 6 13.6 4 2.9 0.014

PREX2 17 38.6 29 21.0 0.018

LRP1B 23 52.3 46 33.3 0.020

HUNK 5 11.4 3 2.2 0.021

ITK 5 11.4 3 2.2 0.021

PRKG2 5 11.4 3 2.2 0.021

WNK2 5 11.4 3 2.2 0.021

PTPRD 15 34.1 25 18.1 0.024

ARID2 9 20.5 11 8.0 0.026

MYO3A 9 20.5 11 8.0 0.026

NTRK3 7 15.9 7 5.1 0.027

CAMK1G 5 11.4 4 2.9 0.039

ACVR1C 7 15.9 8 5.8 0.041

APC 7 15.9 8 5.8 0.041

FLT4 6 13.6 6 4.3 0.041

GCK 6 13.6 6 4.3 0.041

Genes were ordered by p-values.

Supplementary File S1. 632 melanoma candidate genes

This table is an EXCEL file (Supplementary File S1.xlxs)

References

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