Supplementary Methods
Isolation of analytes
Tissue samples were provided by the NCT Heidelberg Tissue Bank in accordance with
its regulations and the approval of the Ethics Committee of Heidelberg University. DNA
and RNA from the tumor specimen and DNA from the blood sample were isolated using
the AllPrep DNA/RNA/Protein Mini Kit (Qiagen, Hilden, Germany), followed by quality
control and quantification using a Qubit 2.0 Fluorometer (Life Technologies, Carlsbad,
CA, USA), a 2200 TapeStation system (Agilent, Santa Clara, CA, USA), and a 2100
Bioanalyzer system (Agilent).
Whole-exome and RNA sequencing
Exome capturing was performed using SureSelect Human All Exon V5+UTRs in-solution
capture reagents (Agilent). Briefly, 1.5 µg genomic DNA were fragmented to 150-200 bp
(paired-end) insert size with a Covaris S2 device (Woburn, MA, USA), and 250 ng of
Illumina adapter-containing libraries were hybridized with exome baits at 65°C for 16 h.
Paired-end sequencing (101 bp) was carried out with a HiSeq 2500 instrument (Illumina,
San Diego, CA, USA) in rapid mode. RNA sequencing libraries were prepared using the
TruSeq RNA Sample Preparation Kit v2 (Illumina). Briefly, mRNA was purified from 1 µg
total RNA using oligo(dT) beads, poly(A)+ RNA was fragmented to 150 bp and
converted into cDNA, and cDNA fragments were end-repaired, adenylated on the 3’ end,
adapter-ligated, and amplified with 12 cycles of PCR. The final libraries were validated
using a Qubit 2.0 Fluorometer (Life Technologies) and a Bioanalyzer 2100 system
(Agilent). Paired-end sequencing (2 x 101 bp) was carried out with a HiSeq 2500
instrument (Illumina) in rapid mode.
Mapping and analysis of whole-exome sequencing data
Reads were mapped to the 1000 Genomes Phase 2 assembly of the human reference
genome (NCBI build 37.1) using BWA (version 0.6.2) with default parameters and
maximum insert size set to 1000 bp.1 BAM files were sorted with SAMtools (version
0.1.19),2 and duplicates were marked with Picard tools (version 1.90). Average target
coverage was 98x for the tumor and 96x for the control. In both, more than 80% of the
targets had a coverage of at least 40x.
For the detection of single-nucleotide variants (SNVs), we applied our in-house
analysis pipeline based on SAMtools mpileup and bcftools with parameter adjustments
1
to allow for calling of somatic variants with heuristic filtering as previously described.3-5
After annotation with RefSeq (version September 2013) using ANNOVAR,6 somatic,
nonsilent coding variants of high confidence were selected. Short insertions and
deletions (indels) were identified using Platypus (version 0.5.2; parameters genIndels=1,
genSNPs=0, ploidy=2, nIndividuals=2) by providing the tumor and control BAM files.7 To
be of high confidence, somatic calls (control genotype 0/0) were required to either have
the Platypus filter flag PASS or pass custom filters allowing for low variant frequency
using a scoring scheme. Candidates with the badReads flag, alleleBias or strandBias if
the variant allele frequency was less than 10%, or more than two of the remaining flags
were discarded. Additionally, combinations of Platypus non-PASS filter flags, bad quality
values, low genotype quality, very low variant counts in the tumor, and presence of
variant reads in the control were not tolerated. Indels were annotated with ANNOVAR,
and somatic high-confidence indels falling into a coding sequence or splice site were
extracted. Copy number variants were analyzed by read depth plots and an in-house
pipeline using the VarScan2 copynumber and copyCaller modules.8 Regions were
filtered for unmappable genomic stretches, merged by requiring at least 70 markers per
called copy number event, and annotated with RefSeq genes using BEDTools.9
Mapping and analysis of RNA sequencing data
RNA sequencing reads were mapped with STAR (version 2.3.0e).10 For building the
index, the 1000 Genomes reference sequence with GENCODE version 17 transcript
annoations were used. For alignment, the following parameters were used:
alignIntronMax
500000,
alignMatesGapMax
500000,
outSAMunmapped
Within,
outFilterMultimapNmax 1, outFilterMismatchNmax 3, outFilterMismatchNoverLmax 0.3,
sjdbOverhang 50, chimSegmentMin 15, chimScoreMin 1, chimScoreJunctionNonGTAG
0, chimJunctionOverhangMin 15. The output was converted to sorted BAM files with
SAMtools,2 and duplicates were marked with Picard tools (version 1.90).
Expression levels were determined per gene and sample as reads per kb of exon
model per million mapped reads, and RefSeq was used as gene model. For each gene,
overlapping annotated exons from all transcript variants were merged into nonredundant
exon units with a custom Perl script. Nonduplicate reads with mapping quality above
zero were counted for all exon units with coverageBed from the BEDtools package.9
Read counts were summarized per gene and divided by the combined length of its exon
units (in kb), and the total number of reads (in millions) was counted by coverageBed.
2
SNVs and indels were annotated with RNA information by generating a pileup of the
DNA variant position in the RNA BAM file with SAMtools.
Western blotting
Monolayers of Plat-E cells and MEFs were overlaid with normal lysis buffer (50 mM
Tris/HCl, pH 7.4; 1% Triton X‐100; 137.5 mM NaCl; 1% glycerine; 1 mM sodium
orthovanadate; 0.5 mM EDTA; 0.01 μg/μl leupeptin; 0.1 μg/μl aprotinin; 1 mM AEBSF)
and detached with a cell scraper. Following centrifugation, supernatants were mixed with
sample buffer (20 v/v% glycerine, 6 v/v% SDS, 6 v/v% ß-mercaptoethanol, 0.06 v/v%
bromophenol blue sodium salt) and subjected to SDS-PAGE. Blotted proteins were
visualized with horseradish peroxidase‐conjugated secondary antibodies (Thermo Fisher
Scientific, Waltham, MA, USA) using the SuperSignal West Femto Maximum Sensitivity
Substrate (Thermo Fisher Scientific) and a Fusion Solo chemiluminescence reader
(Peqlab, Erlangen, Germany).
3
References
1
Li H, Durbin R. Fast and accurate short read alignment with Burrows-Wheeler
transform. Bioinformatics 2009; 25: 1754–1760.
2
Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N et al. The Sequence
Alignment/Map format and SAMtools. Bioinformatics 2009; 25: 2078–2079.
3
Yaktapour N, Meiss F, Mastroianni J, Zenz T, Andrlova H, Mathew NR et al. BRAF
inhibitor-associated ERK activation drives development of chronic lymphocytic
leukemia. J Clin Invest 2014; 124: 5074–5084.
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Jones DTW, Jäger N, Kool M, Zichner T, Hutter B, Sultan M et al. Dissecting the
genomic complexity underlying medulloblastoma. Nature 2012; 488: 100–105.
5
Jones DTW, Hutter B, Jäger N, Korshunov A, Kool M, Warnatz H-J et al. Recurrent
somatic alterations of FGFR1 and NTRK2 in pilocytic astrocytoma. Nat Genet 2013;
45: 927–932.
6
Wang K, Li M, Hakonarson H. ANNOVAR: functional annotation of genetic variants
from high-throughput sequencing data. Nucleic Acids Res 2010; 38: e164.
7
Rimmer A, Phan H, Mathieson I, Iqbal Z, Twigg SRF, WGS500 Consortium et al.
Integrating mapping-, assembly- and haplotype-based approaches for calling
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8
Koboldt DC, Zhang Q, Larson DE, Shen D, McLellan MD, Lin L et al. VarScan 2:
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4
Supplementary Table S1. Coexistence of mutations in RAS familiy members and BRAF
mutants with intermediate activity
BRAF mutation
COSMIC ID
Entity
Sample
HRAS
KRAS
NRAS
PMID
F595L, V600E
F595L
F595La
F595La
F595L
F595L
F595L
F595L
F595L
F595L
COSS1025231
COSS1470210
COSS2097331
COSS2186274
COSS684271
COSS727479
COSS685799
COSS683807
COSS1489268
COSS1718117
Melanoma
Melanoma
UC
UC
CRA
CRA
CRC
CCC
NSCLC
NB
Primary
Primary
Primary
Primary
Primary
Primary
Primary
Primary
Primary
Primary
NA
NA
WT
WT
NA
NA
NA
NA
NA
WT
NA
NA
G12R
G12R
WT
WT
G12D
WT
G12C
NA
NA
WT
WT
WT
NA
NA
NA
NA
WT
NA
15935100
20975100
24121792
24121792
12941809
12438234
12068308
12692057
20881644
22142829
R462I
R462I
COSS685903
COSS980673
CRC
EC
Primary
Primary
NA
NA
NA
WT
NA
NA
12198537
16619251
I463S
COSS685904
CRC
Primary
NA
NA
NA
12198537
G464E
G464E
G464E
G464E
G464E
G464Ea
G464E
G464E
G464E
G464E
G464E
COSS1303034
COSS2163850
COSS2237767
COSS1459722
COSS2141707
COSS724836
COSS1102812
COSS685905
COSS686473
COSS1179048
COSS1879300
Melanoma
Melanoma
Melanoma
HNSCC
HNSCC
OC
OC
CRC
CCC
ACC
BC
Cell line
Cell line
Primary
Primary
Primary
Cell line
Cell line
Primary
Primary
Primary
Primary
NA
NA
NA
NA
WT
WT
NA
NA
NA
NA
WT
NA
NA
NA
WT
NA
G12D
NA
NA
NA
WT
NA
WT
Q61L
WT
NA
NA
WT
NA
NA
NA
WT
NA
19996208
23851445
24714776
20813562
24672248
12068308
18060073
12198537
12692057
19190079
23398044
G464Va
G464V
G464Va
G464V
G464V
COSS1046950
COSS1738950
COSS724835
COSS889335
COSS1781563
BC
BC
CRC
CRC
NSCLC
Cell line
Primary
Cell line
Cell line
Primary
WT
NA
WT
NA
NA
G13D
WT
G13D
NA
NA
WT
NA
WT
NA
NA
17314276
22538770
12068308
14734469
NA
G466A
G466A
G466Aa
G466A
COSS685774
COSS1303054
COSS1646817
COSS1914059
Melanoma
Melanoma
NSCLC
NSCLC
Cell line
Cell line
Primary
Primary
NA
NA
WT
NA
WT
NA
G12C
NA
NA
WT
WT
NA
12068308
19996208
22327622
NA
G469E
G469E
G469E
G469E
G469E
G469E
G469E
G469E
G469E
G469E
G469E
G469E
COSS685351
COSS685798
COSS1651617
COSS886169
COSS1948267
COSS2185962
COSS2121050
COSS2139246
COSS1792997
COSS1799687
COSS683809
COSS1083526
CRC
CRC
CRC
Melanoma
Melanoma
Melanoma
HNSCC
HNSCC
NSCLC
NSCLC
CCC
CC
Primary
Primary
Primary
Primary
Primary
Primary
Primary
Primary
Primary
Primary
Primary
Primary
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
WT
NA
NA
NA
NA
NA
NA
WT
NA
WT
WT
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
14500346
12068308
NA
14722037
23273605
NA
NA
NA
23098378
22649091
12692057
17360030
N581S
N581S
N581S
N581S
N581S
N581S
N581S
N581S
N581S
COSS727252
COSS920871
COSS1166494
COSS1651235
COSS1651277
COSS1651607
COSS1299206
COSS1532583
COSS1746759
CRC
CRC
CRC
CRC
CRC
CRC
Melanoma
Melanoma
Melanoma
Primary
Primary
Primary
Primary
Primary
Primary
Primary
Primary
Primary
NA
NA
NA
NA
NA
NA
NA
NA
NA
Q22K
Q22K
NA
G12D
G12C
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
WT
WT
12438234
NA
18451217
NA
NA
NA
19861964
20925915
22048237
5
N581S
N581S
N581S
N581S
N581S
N581S
N581S
N581S
N581S
N581S
N581S
COSS2013634
COSS2121773
COSS1780107
COSS1780128
COSS1913999
COSS2120683
COSS2120684
COSS1230715
COSS1475020
COSS2157539
COSS2194087
Melanoma
Melanoma
NSCLC
NSCLC
NSCLC
HCC
HCC
MPNST
OC
SIC
RCC
Primary
Primary
Primary
Primary
Primary
Primary
Primary
Primary
Primary
Primary
Primary
WT
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
WT
NA
Q22K
NA
NA
P34L
NA
NA
NA
NA
NA
NA
NA
WT
NA
22842228
NA
NA
NA
NA
NA
NA
19142971
21720365
24797764
NA
L597V
L597Va
L597V
L597V
L597V, V600E
L597V
L597V, V600E
L597V
L597V
COSS686980
COSS724834
COSS1477422
COSS1641854
COSS888999
COSS1664831
COSS1778642
COSS683804
COSS1083527
NSCLC
NSCLC
NSCLC
NSCLC
Melanoma
Melanoma
Melanoma
CCC
CC
Primary
Cell line
Cell line
Primary
Cell line
Primary
Primary
Primary
Primary
NA
WT
NA
NA
NA
NA
NA
NA
NA
WT
WT
WT
NA
NA
NA
NA
WT
WT
WT
Q61K
Q61R
NA
WT
WT
NA
NA
NA
12460918
12068308
20551065
21825258
15048078
21680547
22535154
12692057
17360030
T599I
T599I
T599I, V600E
T599I
T599I, V600E/K
COSS727247
COSS1166491
COSS889188
COSS1238011
COSS1295535
CRC
CRC
Melanoma
Melanoma
BMN
Primary
Primary
Primary
Primary
Primary
NA
NA
NA
NA
NA
G13D
NA
NA
NA
NA
NA
NA
NA
NA
NA
12438234
18451217
15140238
19383316
19752400
Abbreviations: ACC, adrenal cortical carcinoma; BC, breast carcinoma; BMN, benign melanocytic naevus;
CC, cervical carcinoma; CCC, cholangiocellular carcinoma; CRA, colorectal adenoma; CRC, colorectal
carcinoma; EC, endometrial carcinoma; HCC, hepatocellular carcinoma; HNSCC, head and neck squamous
cell carcinoma; MPNST, malignant peripheral nerve-sheath tumor; NB, neuroblastoma; NSCLC, non-small
cell lung carcinoma; OC, ovarian carcinoma; RCC, renal cell carcinoma; SIC, small-intestine carcinoma; UC,
urothelial carcinoma; NA, not available; WT, wildtype; COSMIC ID, Catalogue of Somatic Mutations in
Cancer identifier; PMID, PubMed identifier. aCases with intermediate-activity BRAF mutants in which HRAS,
KRAS, and NRAS were analyzed.
6