Cell Reports, Volume 16

Supplemental Information

Direct Transcriptional Consequences

of Somatic Mutation in Breast Cancer

Adam Shlien, Keiran Raine, Fabio Fuligni, Roland Arnold, Serena Nik-Zainal, SergeDronov, Lira Mamanova, Andrej Rosic, Young Seok Ju, Susanna L. Cooke, ManasaRamakrishna, Elli Papaemmanuil, Helen R. Davies, Patrick S. Tarpey, PeterVan Loo, David C. Wedge, David R. Jones, Sancha Martin, John Marshall, ElizabethAnderson, Claire Hardy, ICGC Breast Cancer Working Group, Oslo Breast Cancer Re-search Consortium,, Violetta Barbashina, Samuel A.J.R. Aparicio, Torill Sauer, ØysteinGarred, Anne Vincent-Salomon, Odette Mariani, Sandrine Boyault, Aquila Fatima, AnitaLangerød, Åke Borg, Gilles Thomas, Andrea L. Richardson, Anne-LiseBørresen-Dale, Kornelia Polyak, Michael R. Stratton, and Peter J. Campbell

! 1!

Supplementary,Methods,

Analysis, of, variant, allele, fraction, differences, between, the, transcriptome, and,

genome,in,TCGA,data,,

!To!validate!our!finding,!we!calculated!differences!in!transcriptional!output!in!TCGA’s!

breast! cancer! cohort.! Aligned! BAM! files! for! 980! breast! cancer! samples!with! both!

RNAESeq! and! exome! sequencing! were! downloaded! from! CGHUB!

(https://cghub.ucsc.edu/)!using!GeneTorrent.! !PCR!duplicates! for!both!exome!and!

transcriptome!were!removed!using!SAMtools.!The!position!of!somatic!mutations,!in!

MAF! file! format,! and! gene! expression! values! (using! the! RSEM! method)! were!

obtained! from! https://tcgaEdata.nci.nih.gov/.! Additional! clinical! covariates! were!

obtained! from! cBioPortal! ! (http://www.cbioportal.org/).! All! putative! mutations!

were! reEannotated!using!Annovar! (release!2013Aug23)! and! all! potential! germline!

variants!were!removed!(present!in!NCBI!dbSNP!Human!build!142).!Finally,!70,071!

exonic/splicing!substitutions!present!in!the!980!RNAESeq!and!WES!paired!samples!

were!considered!for!further!analysis.!Mutations!in!the!5’!or!3’!UTRs!were!excluded.!

Mutated! loci! were! considered! not! expressed,! and! therefore! excluded! from! this!

analysis,! if! the! total! coverage! was! less! than! five! reads,! or! the! number! of! reads!

supporting!the!mutated!base!was!less!than!five!reads.!!

!

These! substitution! mutations! were! evaluated! in! a! number! of! ways,! including! by!

measuring! the! proportion! of! reads! reporting! the! mutation! in! the! transcriptome!

(variant! allele! fraction! or! VAF)! and! subtracting! it! from! the! same!measure! in! the!

genome!(i.e.!VAFdifference!=!VAFtranscriptome!E! !VAFgenome).!We!used!linear!regression!to!

model!the!relationship!between!the!amount!of!ESR1!expressed!by!a!tumor!and!the!

VAFdiff!of!its!mutations.!

!

! 2!

We! classified! TCGA! breast! cancers! into! known! subtypes! (Luminal! B,! Luminal! A,!

HER2Erelated! and! triple! negative)! by! immunohistochemistry! as! per! Blows! et! al!

(PLOS!Med!2010).!!

Estimating,the,excess,of,rearrangements,with,maximum,rank,for,aberrant,

transcription,

We!use!a!maximum!likelihood!approach!to!estimate!the!excess!of!rearrangements!at!

highest! rank.! Basically,! we! allow! the! ranks! to! be! distributed! as! a! multinomial!

process! with! probabilities! of!rank~{π,π,⋯,π,π+τ} ,! where! we! are! interested! in!estimating!!.! It! is! straightforward! to! show! that! the!maximum! likelihood!estimator!for!!!is!given!by:!

! = max 0,! − 1 !! − !!!!!

!! − 1 !! !

where!! !is! the! number! of! samples! (different! ranks)! and!!! !is! the! number! of!rearrangements!garnering!the!!th!rank.!Bootstrapping!of!the!observed!counts!across!all!possible!ranks!was!used!to!estimate!the!95%!confidence! intervals! for!the!point!

estimates.!

!

Detection(of(genomic(rearrangements((

PairedEend! maps! were! generated! using! a! new! inEhouse! algorithm! that! will! be!

published! separately! (J.! Marshall! et! al.,! manuscript! in! preparation).! Briefly,!

discordantly!mapped!read!pairs!were!filtered!against!BWA!read!pileup!loci,!repeat!

features!and!mitochondrial!sequences!in!GRCh37.!Additionally!alternative!mapping!

locations! were! evaluated! to! assess! whether! both! reads! could! be! aligned! to! an!

alternative! location! as! a! concordant! pair.! Remaining! discordant! read! pairs! were!

clustered!to!generate!a!putative!list!of!rearrangements!with!respect!to!the!GRCh37!

reference! genome.! Candidate! rearrangements! found! in! paired! normal! blood! DNA!

analyses,! or! previously! confirmed! by! PCR! to! be! germ! line! in! other! studies,! were!

removed.! These! steps! produced! a! pairedEend!map! cured! from! the!majority! of! the!

artefacts!resulting!from!BWAEmapping!and!from!putative!germ!line!variants.!

! 3!

!

In! this!manuscript! we! only! report! high! confidence! rearrangements! for! which!we!

have! successfully! resolved! the! breakpoint.! To! find! the! breakpoints! we! first!

determined! the! window! surrounding! rearrangements! using! the! average! and!

maximum! insert! size! of! each! BAM! file.!We! then! looked! for! reads!where! one! end!

mapped!within! this!window!and! the! other! end!was!unmapped.! !Unmapped! reads!

were!realigned!to!the!genome!(BLAT,!using!optimised!parameters).!!Realigned!reads!

that!accurately!mapped!within!the!both!windows!of!a!rearrangement!were!grouped!

together,! and! finally! each! putative! breakpoint! was! evaluated! by! measuring! the!

distance!between! the!breakpoint! region!and! the!breakpoint,! and! the!coefficient!of!

variation!of!the!breakpoint!position!themselves!(ideally,!there!is!no!variability!at!the!

position).!!

Validation,of,changes,to,gene,transcript,structure,

We!validated!our!RNAESeq!results!by!using!replicate!RNAs,!comparing!the!junctions!

to!existing!datasets,!using!RNA!pullEdown!sequencing,!and!by!manual!inspection.!

!

Sample! HCC1599! was! run! as! a! technical! replicate.! A! new! library! was! created,!

sequenced! and! analysed! using! the! same! algorithms.! ! One! hundred! percent! of! the!

genomic!rearrangements!causing!an!exon!skip!with!the!highest!rank!were!found!to!

lead!to!the!same!event! in!the!replicate!transcriptome!(5/5).! !Of! the!three!genomic!

rearrangements!involving!two!genes!in!the!same!orientation,!which!were!previously!

found! to! cause! an! expressed! fusion,! two! caused! the! same! event! in! the! replicate!

transcriptome! and! one! was! missed! in! the! replicate.! We! compared! the! inEframe!

fusions!to!those!we!previously!reported!(Stephens!et!al.!Table!3).!Of!the!14!inEframe!

fusions!found!in!both!analyses,!which!had!previously!been!validated!by!RTEPCR,!we!

identified! 10! expressed! fusions! in! the! new! RNAESeq! data! as! well! as! many! other!

others!not!reported!in!the!previous!data!set.!

! 4!

Supplementary,Figures,Legends,

!Supplementary(Figure(1.(RNA(Architect,(a(suite(of(algorithms(for(the(analysis(

of(cancer(RNA<Sequencing.(Related(to(Figure(3.(

(A) Overview!of!RNA!Architect’s!seedEandEextend!and!discordant!pair!algorithm.!

(B) !Statistics!from!a!representative!sample!that!has!been!run!through!this!pipeline.!!

(C) All! samples! sequenced! at! high! depth,! and! there! is! no! association! between!

coverage!and!percentage!of!expressed!mutations.!

(D) Similar!levels!of!expressed!mutation!found!in!TCGA!data.!

!

Supplementary(Figure(2.(Estimating(the(proportion(of(reads(derived(from(the(

tumour(and(the(stromal(cells.(Related(to(Figure(1.(

(A) Comparison!of!variants!from!the!active!and!inactive!X!chromosome.!

(B) Observed! fraction! of! reads! reporting! reference! allele! vs.! the! posterior!

probability!of! the!reference!allele!deriving! from!the!active!X!chromosome.!The!

depth!of!colour!reflects!the!level!of!expression.!

(C) Estimated!distribution!and!95%!posterior!intervals!for!relative!gene!expression!

in!cancer!versus!stromal!cells!for!ER+!and!ERE!breast!cancers.!!

(

Supplementary(Figure(3.((Related(to(Figure(1;(Figure(2.(

(A) Increased! expression! of! the!mutated! allele! in! ERE! as! compared! to! ER+! breast!

cancer!transcriptomes!(plotted!relative!to!the!genome).!

(B) Variant!allele!fraction!in!genome!compared!to!the!transcriptome,!for!all!samples!

including!cell!lines.!

! 5!

(C) Absence!of!negative!selection!in!nonsense!mutations.!Comparison!of!expression!

levels!from!the!organoids!of!normal!breast!epithelium!for!genes!mutated!in!the!

cancer!samples.!!

Supplementary( Figure( 4.( A( recurrent( in<frame( fusion( between( TRMT11( and(

NCOA7*in*two(breast(cancers.(Related(to(Figure(3;(Figure(4.(

A!tandem!duplication!on!chromosome!6!joins!the!5’!end!of!TRMT11!with!the!3’!end!

of!NCOA7.+!In!both!samples!the!fusion!is!inEframe!and!highly!expressed!as!shown!by!

the! numerous! junction! reads! (split! reads)! between! TRMT11+ exon! 11! and!NCOA7+

exon! 13! in! sample! PD4005a,! and! TRMT11! exon! 6! and! NCOA7+ exon! 7! in! sample!

HCC1954.!

+

Supplementary(Figure(5.(Regions(of(local(complexity(in(breast(cancer(sample(

PD4103a.(Related( to( Figure(7.!One!sample’s!regions!of!complexity!are!shown!as!

pairs! of! Circos! plots,! for! the! genome! and! transcriptome.! The! genomic! events! one!

would! predict! to! be! expressed! are! highlighted! (blue! arcs).! The! tumour! does! not!

express!all!of!these!events,!or!multiple!cis+rearrangements!have!been!amalgamated!

and!expressed!as!a! single! transcript! that! combines!genes!only! indirectly! linked! to!

another.!

(

Supplementary(Figure(6.(Compound(event(in(the(gene(MLL3.(Related(to(Figure(

3.((

(A) A!tandem!duplication!in!the!genome!within!the!footprint!of!MLL3,+an!established!

breast! cancer! gene,! results! in! a! complex! aberrant! transcript! involving! the!

reusage!of!exons!and!the!activation!of!an!alternative!donor!site.!The!reads!from!

TopHat! support! junctions! between! the! canonical! exon! edges! (red! arcs)! only!

! 6!

whereas! RNA! Architect! identifies! the! compound! event! (horizontal! lines!

represent!split!reads).!

(B) Aberrant!MLL3+ transcripts.! Shown! are! novel! isoforms! of+MLL3! found! in! TCGA!

breast! cancers! (n=980).! Data! were! reanalysed! and! reprocessed! using! our!

pipeline.! We! compared! each! putative! aberrant! junction! in! MLL3! to! 1,277!

normals! from! 30! tissue! types! and! excluded! anything! found! in! these! samples!

(GTEX).(

(

Supplementary( Figure( 7.( Transcriptional( output( of( ER<positive( and( ER<

negative(breast(cancers.((Related(to(Figure(1.(

(A) The! expression! of! mutations! differs! within! known! molecular! subgroups! of!

breast! cancer.! Samples! were! grouped! using! available! clinical! data!

(Supplementary!Methods),! into!known!molecular! subgroups.!Plotted!on! the!YE

axis!is!the!VAFdiff.!The!pie!charts,!shown!each!subgroup,!depicts!the!percentage!

of!mutations!expressed.!

(B) Differences! in! expression! of! TP53! missense! mutations! between! ER+! and! ERE!

breast!cancers.!

(C) Expression!of!common!mutated!genes!in!EREnegative!and!EREpositive!cancers.!

!

A

Supplementary figure S1

Pre-filter normally mapped reads

Seed-and-extendalgorithm

Build index of known genes

Shatter unmapped reads, align k-mers

Merge and extend k-mers,resolve breakpoints

Perform clean-up of breakpoints

Annotate junction breakpoints

Re-map initially unmapped reads

Discordant pairalgorithm

Merge alignments

Annotate each end of read pair,look for clusters of discordant pairs

Rank putative fusions by number of unique and

multi-mapped reads,consistency of mapping positions

and consistency of discordant read pairs’ orientation

Exon skips

Exon reusages

Alternative donors and acceptors

Early polyadenylation sites

Fusion genes

Fusion genes

B Sequencing depth (read pairs) RNA tumour: 367,423,584DNA tumour: 829,476,568DNA normal: 658,077,733

Exon skipsCanonical: 1944With alt donor or acceptor: 121

PD4107a

Exon reusagesCanonical: 8With alt donor or acceptor: 1

Alternative donors or acceptors: 160

Early poly(A) sitesCoding: 13UTR: 224

FusionsSense: 13Sense with alt donor or acceptor: 4Antisense: 16

D

1.01.52.02.53.03.5

0.00

0.25

0.50

0.75

1.00

log1

0(Co

vera

ge +

1)

Frac

tion

expr

esse

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Breast cancers (n=980)

log1

0(Co

vera

ge +

1)

Frac

tion

expr

esse

d

C

0

1

2

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HCC1143

HCC1187

HCC1395

HCC1599

HCC1937

HCC1954

HCC2157

HCC2218

HCC38

0.00

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1.00

HCC1143

HCC1187

HCC1395

HCC1599

HCC1937

HCC1954

HCC2157

HCC2218

HCC38

PD3851a

PD3904a

PD4005a

PD4006a

PD4085a

PD4086a

PD4088a

PD4103a

PD4107a

PD4109a

PD4115a

PD4116a

PD4120a

PD4248a

PD3851a

PD3904a

PD4005a

PD4006a

PD4085a

PD4086a

PD4088a

PD4103a

PD4107a

PD4109a

PD4115a

PD4116a

PD4120a

PD4248a

Cell lines Primary tumors

C A

T G

XaXi

C A

T G

XaXi

C A

T G

XiXa

C A

T G

XaXi

Cancer cells Stromal cells

mRNA transcripts

A B

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0.0 0.2 0.4 0.6 0.8 1.0

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Observed fraction of reads reporting reference allelePo

ster

ior p

rob

refe

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e al

lele

on

Xa

ExpressionHigh

Low

CPD3904a PD4005a

PD4085a PD4086a

PD4088a PD4107a

PD4115a

0.0 0.2 0.4 0.6 0.8 1.0

PD4116a

0.0 0.2 0.4 0.6 0.8 1.0

PD4248a

Fraction of reads derived from tumour cells

Fraction of reads derived from tumour cells

ER+ breast cancers ER- breast cancers

95% posterior intervalsFitted distribution

Supplementary figure S2

A

B C

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0.00 0.25 0.50 0.75 1.00Variant allele fraction in genome

Varia

nt a

llele

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in tr

ansc

ripto

me

0%

10%

20%

30%

40%

Silent Missense Nonsense

ExpressedIn all organoids

In none of the organoids

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−0.5

0.0

0.5

PD4005a

PD4006a

PD4086a

PD4107a

PD4109a

PD4248a

PD3851a

PD3904a

PD4085a

PD4088a

PD4103a

PD4115a

PD4116a

PD4120a

Varia

nt a

llele

frac

tion

in tr

ansc

ripto

me

rela

tive

to g

enom

e

ER status●

●

−ve+ve

PD4005aPD4006aPD4086aPD4107aPD4109aPD4248a

PD3851aPD3904aPD4085aPD4088aPD4103aPD4115a

PD4116aPD4120aHCC1143HCC1187HCC1395HCC1599

HCC1937HCC1954HCC2157HCC2218HCC38

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Supplementary figure S3

PD4005a

HCC1954

PD4005a

126,333,959-126,337,665 126,210,729-126,241,270

chr6 chr6

NCOA7 exon 13 (NM_001199620)

Junc

tion

read

s in

RNA-

Seq

TRMT11 exon 11 (NM_001031712)

NCOA7 exon 7 (NM_001199620)TRMT11 exon 6 (NM_001031712)

chr6 chr6

126,318,222-126,321,274 126,198,418-126,199,757

In-frame fusion

In-frame fusion

Junc

tion

read

s in

RNA-

Seq

Supplementary figure S4

Genome

Transcriptome

cinegretni_2 STAT

1LR

P1B

ROBO

23_

inte

rgen

icAT

XN7

PDZR

N3CG

GBP1

EPHA

6RO

BO1

LSAM

PABC

E1LA

RP1B

MAPK10PTPN13

INPP4B

C4orf43

USO1

RUFY3

MARCH1

4_intergenic

RXFP1

EDIL3

5_intergenic

NDUFAF2

CDH12GALNT10

7_intergenic

AUTS2ZNF804BKIAA0196NECAB1

ENSG00000235517ADAM18CHD7TACC18_intergenicFER1L6SLC20A2NBNMTSS1NSMCE2UNC5D

9_intergenic

PTPRDITIH5

10_intergenic

FAM107B

KIAA1217

GPR158

ITIH2

CAMK1D

PLXDC2

ARMC4

MLLT10

PCDH15

ERCC6U

SP6NL

ST8SIA6C10orf112CH

KA2

NBTP

S

ACER

3BR

MS1

GD

PD4PC

ANO

1

CCND

1

C2CD

3

C11o

rf80

ORAO

V1

11_i

nter

geni

c

PAK1

SHANK2

XRRA1

MRPL21GLT

P

MDM1FICDHELBGRIP1ANKRD13APTHLHCDK17SRGAP1CCDC38TRHDELGR512_intergenicC12orf66

BEST3PTPRB

TSPAN8SSH1

16_intergenic

17_intergenicZNF28

NCOA3

LAMA5

ASXL1

CABLES2

BCAS1

EFCAB8

DOK5

20_intergenic

SULF2

UBE2G2

DSCAM

SLC37A1B3GALT5

HLCSPCNT

C21orf29SIM

2RUNX1

TMPRSS3

DOPEY2

21_intergenicGK

IL1RAPL1D

MD

X_intergenicKD

M6A

cinegretni_2 STAT

1LR

P1B

ROBO

23_

inte

rgen

icAT

XN7

PDZR

N3CG

GBP1

EPHA

6RO

BO1

LSAM

PABC

E1LA

RP1B

MAPK10PTPN13

INPP4B

C4orf43

USO1

RUFY3

MARCH1

4_intergenic

RXFP1

EDIL3

5_intergenic

NDUFAF2

CDH12GALNT10

7_intergenic

AUTS2ZNF804BKIAA0196NECAB1

ENSG00000235517ADAM18CHD7TACC18_intergenicFER1L6SLC20A2NBNMTSS1NSMCE2UNC5D

9_intergenic

PTPRDITIH5

10_intergenic

FAM107B

KIAA1217

GPR158

ITIH2

CAMK1D

PLXDC2

ARMC4

MLLT10

PCDH15

ERCC6U

SP6NL

ST8SIA6C10orf112CH

KA2

NBTP

S

ACER

3BR

MS1

GD

PD4PC

ANO

1

CCND

1

C2CD

3

C11o

rf80

ORAO

V1

11_i

nter

geni

c

PAK1

SHANK2

XRRA1

MRPL21GLT

P

MDM1FICDHELBGRIP1ANKRD13APTHLHCDK17SRGAP1CCDC38TRHDELGR512_intergenicC12orf66

BEST3PTPRB

TSPAN8SSH1

16_intergenic

17_intergenicZNF28

NCOA3

LAMA5

ASXL1

CABLES2

BCAS1

EFCAB8

DOK5

20_intergenic

SULF2

UBE2G2

DSCAM

SLC37A1B3GALT5

HLCSPCNT

C21orf29SIM

2RUNX1

TMPRSS3

DOPEY2

21_intergenicGK

IL1RAPL1D

MD

X_intergenicKD

M6A

PD4103a

Supplementary figure S5

Fusion to antisenseFusion

Alt donor / acceptorEarly polyA siteExon reusageExon skip

Complex rearrangement predicted to cause a fusion, exon skip or reusage

Complex rearrangementGenome arcs

Transcriptome arcs

DN

A re

arra

ngem

ents

151,874 kb 151,878 kb 151,882 kb 151,886 kb 151,890 kb 151,894 kb

21 kb

Internal duplication

Exon reusage with cryptic donor

MLL3 (KMT2C)

RNA

junc

tions

TopHat

Architect

Supplementary figure S6

151,900 kb 152,000 kb 152,100 kb304 kb

RNA

junc

tions

MLL3 (KMT2C)

A

B

Supplementary figure S7

0.00

0.25

0.50

0.75

Luminal B(7,349 muts. in 101 tumors)

Luminal A(16,652 muts. in

357 tumors)

HER2-related(1,034 muts. in 27 tumors)

Triple negative(5,597 muts. in

86 tumors)

VAF

Diff

eren

ceA

B

Expr

esse

d m

uts.

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0.00

0.25

0.50

0.75

1.00

DNA RNADNA RNA

Prop

ortio

n of

rea

ds

repo

rtin

g m

ut.

ER+ (49 tumors)

TP53 missense mutations

ER- (42 tumors)

● ●

●

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0

5

10

15

0 5 10 15

●●●●●

●●●●●

●●●

APH1ACDH1CXorf40ADDX49IGSF8

ITCHPIK3CAPSMA5PSMA7PTEN

TCTN2TP53YAP1 E

xpre

ssio

n o

f mut

ated

gen

es

in E

R- c

ance

rs (l

og2(

TPM

))

Expression of mutated genes in ER+ cancers (log2(TPM))

C