Figure S1. The correlations of TMB value between panel sequencing and WES in published databases.

(A) TCGA-LUAD, (B) TCGA-LUSC, (C) Database from Rizvi H, et al. J Clin Oncol, 2018;36.

Abbreviations: TMB, tumor mutational burden; TCGA, The Cancer Genome Atlas; LUAD, lung

adenocarcinoma; LUSC, lung squamous cell carcinoma; WES, wholeexome sequencing

A B

C

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Figure S2. The comparison of multi-region tTMB among different NSCLC subtypes.

Abbreviations: TMB, tumor mutational burden; tTMB, tissue TMB

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Figure S3. The comparisons and overlaps of tumor-derived mutational profiles among tumor tissues in each region and the corresponding ctDNA.

The P0XX was the patient No. shown at the top. Each tumor region (T1, T2, T3…) with plasma (P) were arranged in the x axis. ctDNA was isolated from plasma (more details in Supplementary Methods). Right y axis displayed tumor-derived mutational profiles in detail. The detected mutations were shown in red, while undetected cases were shown in gray.

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T1

T1 T1

T2 P022 P027P028 P029 T2 T2 T3 T3 P008 P016P017P020 T3 P001 P004P005P006 T4 P T4 T5 TRPC5.p PTPRD.p MED12.p CCND2.p NO HD SLC17A6.p OR2T4.p NTRK3.p IGF1R.c.3297.1G.T TBP.p NO OCA2.p TNR.p UNC13D.c.684.2A.C XRCC1.c.1482.1G.T HGF CDH23.p RET SLC4A10.p COL25A1.p AKR1B10.p TP53.p KDR.p PDGFRA.p EGFR.p FO CDH23.c.754.2A.T HGF OR5L2.p SETD2.p CDKN2A.p F OR5L2.p DNMT3A.p ALK.p ABCC11.p TCP11.p FGFR1.p OR2T4.p ERBB4.p NO FGFR4.p HECTD4.p SMARCA4.p CDH23.p MTTP.p HD DUO F OR2T4.p CDH23.p ERBB2.p HCLS1.p F SMARCA4.p KRAS.p VEGF EPHA3.p CD97.p DDR2.p EPHA3.c.1307.1G.T PIK3R2.p F CSMD3.p RICT A A A AM5C.p T1.p T1.p T1.p XL2.p A A TCH4.p TCH4.p TCH1.p .p .p .p C4.p C4.p X1.p OR.p T5 A.p .E235K .E152. .Q89H .R556S .V64. .L906P

.Q72. T6 .M1533I .A3666T .V2936. .Q38. .K156Rfs .S385C .W532L .G12V .R746L .Y1110C .Q344. .Q43H .T57N .L220. .R51K .R484S .L58I .Y136. .E394D .M86I .L801F .E803. .V740L .V1734E .G565C .G252C .F895L .R816L .P291Q .Q471K .G117V .K1071N .E645D .G1293A .R1219Q .D991H .L20M .G363C .H775N .A3V .L958Q .G4219V .G101V .G1081C .L900I .R2327W .G800R .P6S .L416F .S416Ffs .R375G .R269S .K455M .E1242G

P EML4.ALK T7 .6 .27

MLH1.p CTNNA2.p ALK.p SMARCA4.p KRAS.p OR4A15.p F MT P AM5C.p OR.p PPEF1.p EGFR.p PTCH2.p CSMD3.p A MLL4.p A FMN2.p TP53.p MTTP.p EPHA5.p INHBA.p ARHGEF1.p NO POLR3B.c.1102.3_1102.2delT .M1478T TG9B.p TR.p .G517R .G12D .N1264S TCH1.p T1 .P690T .L83M .R125Q .Q416E .R234L .A503S .D1842E .E1640K .S383I .E746_A750del .E154K .R298C .R348Q .E854K .F214C .H1455Y

T2 .Q2361. .S25F

T3

T4 T1

T5 T2 A

T3 T6 T1

T4 T7 T2 T5 T8

T6 T3 P T7 BRD2.p NF1.p A COL6A5.p SMO.p MLL4.p CD74.R NF1.p CDK13.p EPHA3.p RAF1.p PRKAA1.p ALK.p BRCA2.p KCNH6.p EPHA3.p ABCC11.p NO ST MLL3.p ABL2.p MLL3.p BRCA2.p MLL4.p MDM4.p FGFR1.p NF2.c.1575.1G.C MAP3K1.p F MAP3K1.p MLL3.p KIAA0319.p C11orf30.p MITF ASTK.p TM.p P A

TCH2.p T8 T3.p .p .Q83. .D1849N .E717K .A2346G .Q30. .E779K .E398K .D131H .S1228. .Q1211. .D168H .D3152N .E503Q .K127N OS1 EPHA5.p MLL3.p OR5L2.p TP53.p ANKRD27.p TER NTRK3.p TSC1.p TENM3.p F MORC1.p .E625D .R149G P .K530N .S1066. .F114V .R330K .Q139. A .D1990H .E2599K .D673H .M1319V T1.p .E1169K .S5R .P981S .K953R .E494Q .Q728. .D909Y T .c..58.u3403C.A STK11.c.456_464.6del NF1.p NO NO BRAF AR.p FBXW7.p TP53.p CR TER AR.p CRKL.p EPHA5.p OR2T4.p OR4A15.p GNAS.p TP53.p R CD274.p RET CCNE1.p FL .R4481L .V134A .Q560Sfs .R779. OS1.p T3.p .C260. .D208E .S992I YBG3.p .S44I TCH2.p TCH2.p .G97V .p T .Q71R .Q74R .c..58.u2712G.T .p .G744V .E713D .V350G .Y874C .H76Ifs .R119L .G469V .P231S .V1175L .S455Y .L214. .I229F .D180Y .R385M .R473G .P86H .53 .K2356Rfs .L1844V .R1578C .8 T1 T1 T2

T2 .3 T3 P030 P031 P035 P036 T4 T3 T1 T5 T4 T1 T6 T5 T7 T6 T2 T8 T2 P T7

MDM2.gain MET NO ALK.p T8 T3 T3

TCH1.p P .p .A892T .Y1021S KRAS.p HCN1.p OR4A15.p GA DNMT3A.p FL TP53.p NO NO EMID2.p T4

T1.p T4 T TCH4.c.2022.1G.T TCH2.p .N1603T A3.c.1050.2T

.S530P T5 .A99V .G12V .R593Q .L96Q .C217F .G523V .G298W P T6 CCNE1.gain POM121L12.p MSH3.p ERBB4.p TP53.p ERBB2.p NF1.p FL DDR2.p SPT .A

P T1.p A1.p MET CDH18.p FGFR1.p TP53.p MYD88.p MET NA TRIM58.p ANKRD36B.p OR6F1.p PTPRD.p EPHA5.p TP53.p OR4C6.p OR4C6.p EPHA5.p AR.p SLCO5A1.p PDGFRB.p CDH23.p AR.p ERBB4.p .A1228T .K37E .H193Y .R478C .A51.12.9. V3.p .S444C .R484K .G746delinsVC .c.2941.1G.T .p .G260V .I304V .Y989N .K100N .R234S

T1 .D414V .D269Y .R448W .G664. .P561L .I850T .I743F .P309Lfs .L265P .L280V .M279I .V834L .D321E T1 .D11E .P725R .V677L

T2 .I480M

T3 T2 .7 T4 T5 T3 T6 T1 T1 P002 T1 T7 T2 P T2 T3 T4 T2

C1S.p HCN1.p TP53.p INHBA.p CDH23.p T4 T3 P T3 EGFR p.E746_A750del .E98.

.R241K P .R593. .R379W .R2066W T4 T4 TMTC4.c.1586.1G.A R AXL.p CTNNB1.p ENPP2.p TP53.p R XRCC1.p TP53.p EPHA2.p TESC.p NF1.p CNO MLL3.p RB1.p A DDR2.p R W DDR2.p ARHGAP40.p PTEN.p FBXW7.p RB1.p HD TM.p OCK1.p OS1.p OBO3.p T5 AS.c.1453.1G.T P P033P034P037 A C4.p T3.p .A2028S .V406L .T601Ifs .T601Ifs .R250Q TP53.p MAEL.c.703.1G.C APC.p MLH1.p EGFR.gain EGFR.p A .C199F .G227. .R4478P .R59L .V166G .S852R .P384H .R128H T6 CIN1.p .R446. .N71Y .S735R .K544N .I232M .V1127G .W244. .S307I .I507. P021P030P025 .E2184K P007P009P015P018 .Q105_S110delinsP .P447T .7.1 .7 .L658V

P .L858R .K1047N T1 TP53.p AXL.p ERBB2.p FGFR2.p SMARCA4.p F EZH2.p THBS2.p MLL4.p GFRAL.p F ERBB4.p MT DNMT3A.p COL25A1.p MLL3.p MED12.p RICT PTPRD.p GNAS.p F FGFR4.p MLL4.p CNTN5.p MLL.p MYC.p R ABCC11.p R IGSF9.c.1198.1G.A CNTNAP5.p GNAS.p A A A OS1.p OS1.p P032 T1.c.3629_3642.5del T1.p T2 T1.p OR.p OR.p .E281G T3 .P36L .K158E .P381R .D1204V .S201C .V169L .A221P .C1325F .T244M .R2039P .L567M .R524S .S110C .E742K .C835Y .C809F .R196L .E580G .G1293V .H206Q .T176N .Q2146.5.4. .D848N

T4 .I188F .C609F .G332R

T1 .G382Dfs .Q909L P .Q459_K461del

NTRK3.p TP53.p TP53.p T2 T1

T3 .48 .V177Dfs .V177L

.P562Q T4 T2 T5

T6 T1 T3

T7 T2 T4 T8 T3 T1 P T4 P T2 BRCA1.p TMEM247.p NO APC.p CCND1.gain OR4C6.p TP53.p NO FGFR3.p NO TGFBR1.p MLL3.p NTRK3.p MPL.p GNAS.p GP T5 PDGFRA.gain KIT KDR.gain NTRK2.p KDR.p F PRKCD.p COL6A6.p DNMT3A.p XRCC1.p STK11.p EPOR.p TP53.p KRAS.p A TCH2.p TCH2.p TCH4.p A T3 TCH2.p T1.p .gain .Q1469. .R43. .C203F .G3106R T6 .S532C .S410R .S249F .C166F .K1233. .C205F .V4575E

T4 .Y163C .G12C .G301R .R45P .P2463S .H2041R .P445H .I238F .R413I .P383L .E455Q .G86. .G210V

T7 .P1542S P .F563I

EML4.ALK KRAS.p J J NFE2L2.p MLL3.p P AK3.p AK3.p FBXW7.p ABL1.p TNN.p MLH1.p C1S.p KRAS.p MT DOCK3.p SMARCA4.p MT GAB2.p GPR114.p FGFR3.p FO R CDK13.p MITF .P893L .R403H OS1.p .C344S .G13D XL2.p OR.p OR.p .E79Q .p .N233Tfs .P680Q .A285Pfs .T993A .Q149Rfs .Q562Sfs .G666Vfs .G12V

.N2494Ifs .F191Lfs T1 .P285Rfs .R1317Efs .Y673Hfs .W486Gfs .L1854H T1 .M34T T1

.E665_E666delinsD. T2 T2 P003 .6 .12 T3 .20 .172 .16 .11 T3

T2 .74 .71 .5 .12 .39 T4 T5 T4 T3 T6 T5 T1 P T4 KEAP1.p T6 T2 P T3 T5 EZH2.p MYD88.p PIK3CA.p BAX.p T1 .E244K T4 T6

T2 .E41G

T5 .S40Ifs P .L265P T6 T3 .R108H OR2T4.p EPHA2.p KEAP1.p ERBB4.p TP53.p CNO P CCNE1.p FUNDC1.p NF1.p ELA ALB2.p .17 P T4 ERBB2.gain CCND1.gain CDH23.p ERBB2.p TP53.p VL3.p T3.p .I177T .R234H

.S631P T5 .A30D .A40S .V442A .P572L .P269Afs .R27H .A18Rfs .N200. .E130Q .E3319K .Y772_A775dup T6 T1 T1

.34 T2 .15 T7

T3 P019 T2 T8 T4 T5 T3 P T6 T4 TSC1.c.2623.2_2623.1insA EGFR.p TMEM247.p GNA A SLC25A30.p CDK4.gain EGFR.gain TP53.p T7 T1 URKA.p T8 P Q.c.736.2_736.1insA

P PRSS1.p FL TP53.c.443.2A.C EGFR.p V .N272Kfs A

T2 .L858R T4.p V1.p T1 RB1.c.1696.1G.C NO EGFR.p TP53.p BRAF LCT .K143E P038 .Q152_Q153ins TCH2.p .W266Gfs .A601V .p T2 T3 .R548W .L858R .c.2128.2_2128delA .F314Lfs .E32D .W52. .33 .L858R T3 T4 .E301K

P .5 T5 .7 MT SPT TP53.p EGFR.p OR.p A1.p T6

.H140Y T1 .L858R .A1922V .N878D GA T7 T2 T8 T1 T1 T3 T2 P026 P T3 T2 T4 RB1.p MAP2K1.p NF1.p CDK12.p ERBB2.c.2086.1G.A RPS6KB1.p TP53.p KEAP1.p KEAP1.p SMARCA4.p EPOR.p MLL4.p CHEK2.p SETD2.p SETD2.p FGFR3.p F PDGFRA.p TER ESR1.p CCND3.p MLL3.p EGFR.p HGF TSC1.p GNA MED12.p ERBB2.gain CDK12.gain FGFR2.p MLL.p NO MCL1.p HRAS.c..53.1G.T CCND1.p OR6F1.p ERBB3.p C1S.p HD T4 A T3 T1.p A TCH2.p T5

.p T5 T C1.p Q.p .c..58.u1680C.A .L1183V .Q257. .D649H .Q318H .F99L EPHA3.p CDK13.p EGFR.p MSH6.p U2AF1.p CHI3L1.p EGFR.p P RB1.p .P1877Lfs .R209L .P1124R .H4429L .A566V .D332Y .G59_G62del .E45K .S176F

.G46W P .E850K .D631N .T160I .S256W .R601L .D526Y .S612F .C740G .Q239E .G280A .L106V .S2163F .A266V .E76Q T6 .R201C .E2143Q ALK.p CTNNB1.p EGFR.p CTNNB1.p MAP2K1.p TP53.p .P698Q .A433Lfs .A348T .Y439. .A750P .S1340C .L747_E749del .S34F .K852Nfs .T519M .G37S P .20 .P496S .M345Nfs .24 .E746_A750del .23 RET PTPRD.p SEPT12.p OR4A15.p NO POM121L12.p TP53.p ALK.p NA .L375P .Q72E .S37F V3.p TCH1.p .p .G727. .A195V .39 .R209L .A503P .T799A .R266Q .V222I .E1583Sfs .R49P .33