Supplementary Table 1 Genes included in the panel

Gene / FOXO3 / NOTCH2
ABL1 / FUBP1 / NRAS
ACVR1 / GABRA6 / NTRK2
AKT1 / GNA11 / PCDH8
AKT2 / GNAQ / PDGFRA
AKT3 / GNAS / PIK3C2G
ALK / H2AFX / PIK3CA
APC / H3F3A / PIK3R1
ARID1A / HDAC2 / PPM1D
ARID1B / HIST1H3B / PRKAR1A
ARID2 / HIST1H3C / PTCH1
ATM / HNF1A / PTCH2
ATR / HRAS / PTEN
ATRX / IDH1 / PTPN11
BCOR / IDH2 / Rad50
BRAF / IDO2 / RAF1
BRCA1 / JAK2 / RB1
BRCA2 / JAK3 / RET
BRPF1 / KDM6A / SETD2
BRPF3 / KDR / SMAD4
C11ORF95 / KIAA0182 / SMARCA2
CCND1 / KIT / SMARCA4
CCND2 / KLF4 / SMARCB1
CDH1 / KLK1 / SMARCD1
CDK4 / KRAS / SMARCD2
CDK6 / LDB1 / SMARCE1
CDKN2A / LZTR1 / SMO
CDKN2B / MDM2 / STAG2
CDKN2C / MDM4 / SUFU
CHEK2 / MET / TBR1
CHEK2 / MGMT / TCF4
CIC / MLH1 / TERT
CREBBP / MLL2 / TP53
CSF1R / MPL / TRAF7
CTNNB1 / MRE11A / TSC1
D2HGDH / MSH2 / TSC2
DAXX / MSH6 / VHL
DDX3X / MYB
DICER1 / MYBL1
EGFR / MYC
EZH2 / MYCN
FBXW7 / MYL1
FGFR1 / NBN
FGFR2 / NDRG2
FGFR3 / NF1
FGFR4 / NF2
FLT3 / NOTCH1

Supplementary Table 2 Excerpt from De-Fuse fusion detection

De-Fuse output contains the exact break positions within the genes involved in a fusion.

Supplementary Table 3 Molecular targets identified by panel sequencing within the prospective target discovery group (47 glioblastomas, 10 pilocytic astrocytomas, 14 medulloblastomas), and respective recruiting clinical studies (some cases harbor more than one target)

Entity / Aberration / Frequency / Study / Comment / Drug
GBM / EGFR amp/activation / 16/47
(34%) / NCT02365662
NCT01953926
NCT02465060 / ABBV-221
Neratinib
AZD9291
GBM / PTEN / 15/47
(32%) / NCT01870726
NCT02449538
NCT02233049
NCT01458067
NCT02286687
NCT01470209 / Refractory GBM
Primarily DIPG
Relapsed/refractory solid tumors
Relapsed/refractory solid tumors
Refractory solid tumors / INC280 & BKM120
everolimus
erlotinib, everolimus or dasatinib
GSK2636771
BMN673
BKM120 & everolimus
GBM / PIK3CA / 3/47
(6.3%) / NCT02449538
NCT01470209 / Refractory cases
Refractory solid tumors / everolimus
BKM120everolimus
GBM / PDGFRA amp / 5/47
(11%) / NCT02029001
NCT02219711 / (phase I) / Nilotinib or Pazopanib
MGCD516
GBM / CDK4 amp / 2/47
(4.3%) / NCT02187783
NCT02065063
NCT02530320 / (primarily oligodendroglial tumors) / LEE011
TrametinibPalbociclib
Palbociclib
GBM / MSH6 / 2/47
(4.3%) / NCT01876511 / Microsatellite-unstable cases / MK-3475
LGG / BRAF/KIAA1549 Fusion, BRAFV600E / 7/10
(70%) / NCT01386450
NCT01089101
NCT02285439 / AZD6244
Selumetinib
MEK162
LGG / FGFR1 / 2/10
(20%) / NCT01975701 / BGJ398
MBIV / PTCH1, SMO / 3/14
(21.4%) / NCT01878617
NCT01601184 / Vismodegib
Vismodegib +/-TMZ
MBIV / PIK3CA / 2/14
(14.3%) / NCT02449538
NCT01470209 / Refractory cases
Refractory solid tumors / everolimus
BKM120everolimus

Supplementary Table 4Cost for NGS

Enrichment kit (Agilent SureSelect) / 260 €
Sequencing Flow Cell/Cartridge (IlluminaNextSeq 500) / 110 €
Other consumables (QC, shearing) / 15 €
385 €

Cost per sample based on list prices (Oct 2015), assuming a sequencing run of 12 samples with approximately 500x average coverage. Discounts may apply for bulk orders. Low DNA quality may reduce the feasible amount of samples per flow cell and/or coverage.

Supplementary Figure 1 Workflow

Panel-Sequencing workflow from tissue to report and respective consumables and devices

Supplementary Figure 2 Representative copy-number-plots derived from NGS data

aAmplification of EGFR

bAmplification of PDGFRA

cDetection of EGFRvIII

Assessment of EGFR mapped read counts in 11 EGFR amplified glioblastoma indicates deletion of exon 2-7 representing EGFRvIII in 4/11 samples.

Supplementary Figure 3Detection of low abundance variants in tumor DNA diluted in germline DNA (1:100)

A 2 bp deletion within ATRX (horizontal black bar) is illustrated in DNA of a diffuse astrocytoma sample after diluting the tumor DNA in wild-type germline DNA by a factor of 1:100.

Supplementary Figure 4

Sample report with copy-number-plot and SNVs derived from panel sequencing.