Download Whole Genome Low Pass Sequencing

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SUPPLEMENTARY INFORMATION
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METHODS
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Tumor sample collection and cell culture
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Resected brain tumor specimens were collected at Henry Ford Hospital (Detroit, MI) with written
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informed consent from patients, under a protocol approved by the Henry Ford Hospital
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Institutional Review Board, and graded pathologically according to the WHO criteria. A portion
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of each tumor specimen was snap frozen and stored in liquid nitrogen. An adjacent portion was
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used for cell culture. Tumors are dissociated enzymatically and neurospheres enriched in
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cancer stem-like cells (CSC) were cultured, as described in detail 1,2. Neurosphere cultures
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were serially passaged in vitro.No mycoplasma contamination was identified in the subset of
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samples tested. Cells with passages between 7 and 18 were used for mouse implants and
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molecular analysis, except for those designed “high passage”, where passage 40 was used.
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Patient derived xenografts (PDX)
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Orthotopic xenografts: Following IACUC guidelines in an institutionally approved animal use
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protocol, GBM neurosphere cell suspensions were implanted into 8-week old female nude mice
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(NCRNU, Taconic Farms) as described 3. A minimum of 8 mice were implanted with each
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neurosphere line. Animals were anesthetized with a mixture of ketamine and xylazine.
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Dissociated neurosphere cells (3x105) were injected using a Hamilton syringe at a defined
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intracranial location: AP+1.0, ML+2.5, DV-3.0. Animals were monitored daily by an observer
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blinded to the group allocation and sacrificed upon first signs of neurological deficit or weight
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loss greater than 20%. Brains were harvested, placed in a coronal matrix for 2 mm sections,
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with the first cut across the implant site. Brain sections were alternately frozen in dry ice and
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embedded in OCT for storage at -80oC, or formalin fixed and paraffin embedded (FFPE).
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Subcutaneous xenografts: Dissociated neurosphere cells (1x106) were injected in the flank of
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nude mice. Animals were sacrificed and tumors excised when diameter reached 10 mm.
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Drug Treatment: HF3077 PDXs were treated with capmatinib (purchased from Matrix Scientific
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Products (Columbia, SC)) suspensions in 0.5% methylcellulose/0.1% Tween 80 were prepared
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every week and administered by oral gavage using a 20g x1.5” gavage needle (Cadence) at a
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dose of 30 mg/kg once a day (5 days/week) until the end of the study. Control animals received
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vehicle only mock gavage. Forty-five days after implant, animals were randomized to control or
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treatment groups. Each mouse was followed until death with no censoring and mean survival
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differences were estimated using a t-distribution to estimate 95% confidence intervals. With a
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sample size of 9 mice per group, a two-sided 95% confidence interval for the difference in mean
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survival would extend 0.92SD from the observed difference in mean survival, assuming the CI is
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based on large sample z statistic. Equivalently we expected 80% power to detect a difference in
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mean survival of 1.4SD, for the common standard deviation, when n=9 animals per group and
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alpha=0.05. Animals were monitored daily and sacrificed upon first signs of neurological deficit
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or weight loss greater than 20%. Control animals were administered vehicle. Kaplan-Meier
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Survival curves were compared by log-rank test.
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To evaluate brain penetrance of capmatinib, 2h after administration of the last capmatinib 30
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mg/kg dose, blood samples were drawn, animals were sacrificed and brains were harvested
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and 2mm coronal sections were frozen in OCT. Tumor tissue was dissected from the frozen
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blocks. Capmatinib concentration in homogenized tumor tissue and plasma was determined for
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3 treated animals and one control was quantified by LC-MS/MS .
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Xenograft tumor macrodissection of frozen tissue
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Brain samples of 3 randomly selected animals per xenograft line were used. Frozen 2mm
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coronal sections were transferred to a cryostat (Cryotome E, ThermoElectronCorporation) set to
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-16oC. Six m sections were cut and stained with hematoxylin, to locate the tumor. Tumor tissue
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was excised from the frozen block with a scalpel into a pre-chilled microtube and stored at -
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80oC.
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Nucleic Acids isolation
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Genomic DNA was isolated from frozen tumor samples, macrodissected xenograft tumor (3
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biological replicates), and neurosphere cultures using QIAamp DNA mini Kit (Qiagen #51304),
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with on column RNase A digestion, following manufacturer instructions. DNA was isolated from
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blood using DNA QIAamp Blood kit (Qiagen).
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Total RNA was extracted from frozen tumor samples, macrodissected xenograft tumor (3
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biological replicates), and neurosphere culture using MirVana (Ambion # AM1560), followed by
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DNAse treatment using DNA-free (Ambion AM1906).
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Fluorescence in situ hybridization (FISH)
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FISH on matching tumor samples/neurospheres/PDX: FISH probes were prepared from purified
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BAC clones (BACPAC Resource Center, https://bacpacresources.org). Probes were labeled
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with Orange-dUTP or with Green-dUTP (Abbott Molecular Inc., Abbott Park, IL), by nick
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translation.
Locus
Human BAC Clones:
12q13.3 - q14.1 (CDK4 gene)
7p11.2 (EGFR gene)
8q24.21 (MYC gene)
7q31 (MET gene)
4q12 (PDGFRA gene)
4q11 (Ch. 4 control)
7q11.22, (Ch 7 control)
8q11.21 (Ch. 8 control)
RP11-181L23, RP11-571M6, RP11-277A02
RP11-708P5, CTD-2026N22, RP11-148P17
CTD-3056O22
RP11-95I20, RP11-564A14, RP11-39K12
RP11-58C6 and RP11-977G3
RP11-365H22
RP11-747K2, RP11-668K3
CH17-311E13 and CH17-425G9
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Metaphase slides were prepared from neurosphere cell cultures that were harvested and fixed in
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methanol:acetic acid (3:1), according to standard cytogenetic procedures. Tumor touch
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preparations were prepared by imprinting thawed tumor tissue onto positively-charged glass
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slides and fixing them in methanol:acetic acid (3:1) for 30 min then air-dried. Frozen tumor and
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macrodissected xenograft tumor samples were prepared as described 4. The FISH probes were
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denatured at 75 °C for 5 min and held at 37 °C for 10-30 min until 10 ul of probe was applied to
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each sample slide. Slides were coverslipped and hybridized overnight at 37 °C in the
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ThermoBrite hybridization system (Abbott Molecular Inc.). The posthybridization wash was with
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2X SSC/0.2% TWEEN 20 at 73 °C for 3 min followed by a brief water rinse. Slides were air-dried
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and then counterstained with VECTASHIELD mounting medium with 4'-6-diamidino-2-
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phenylindole (DAPI) (Vector Laboratories Inc., Burlingame, CA).
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Image acquisition was performed at 1000x system magnification with a COOL-1300
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SpectraCube camera (Applied Spectral Imaging-ASI, Vista, CA) mounted on an Olympus BX43
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microscope. Images were analyzed using FISHView v7 software (ASI) and 100 - 200
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interphase nuclei were scored for each sample in addition to analysis of 50 - 100 metaphase
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spreads for each cell line.
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FISH on paired primary/recurrent FFPE gliomas: Fluorescence in situ assay was performed
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using RPS6/Con 9, CDK4/Con 12, EGFR/con 7, MYC/ con 8, PDGFRA/con 4, C-Met/con 7,
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TERT/Con 5 FISH probes from Empire Genomics (Buffalo, N.Y.). The slides were hybridized
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with the FISH probes according to the manufacturer's instructions with slight modifications.
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The slides were then examined under fluorescence microscope (Nikon 80i) equipped with
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multiple filters and signals were manually counted in 50 cells for each slide.
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Immunohistochemistry
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Sections of formalin fixed, paraffin embedded human glioma surgical samples, tumor
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xenografts, or multicellular spheroids were deparaffinized with xylene and rehydrated through
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graded alcohol into in phosphate buffered saline. Antigens were unmasked by 10 min incubation
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in boiling in citrate buffer and sections stained with anti-Met rabbit monoclonal antibody (D1C2)
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(Cell signaling #8198) or anti-phospho-Met (Tyr1234/1235) rabbit monoclonal antibody (D26)
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(Cell signaling #3077) and visualized with Betazoid DAB (Biocare BDB2004) and counterstained
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with Envision Flex Hematoxylin (Dako K8008). Images were captured using a Eclipse E800M
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microscope equipped with a Nikon DS-Fi2 color digital camera (Nikon).
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Reverse Transcription and PCR
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cDNA was prepared from 1 g DNAseI-treated total RNA isolated from tumor, neurosphere and
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xenografts using Superscript III Reverse Transcriptase and oligo dT (Thermo Fisher Scientific).
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cDNA was used as a template for PCR reaction in a iCycler instrument (BioRad), using
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Platinum Taq DNA Polymerase (Thermo Fisher Scientific) and the following oligos:
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Human MET: exon 2 forward (M2F): 5’ AGCAATGGGGAGTGTAAAGAGG and exon 8 reverse
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(M8R): 5‘ GTAAGTAAAGTGCCACCAGCC
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Human CAPZA2 exon 1 forward (C1F): 5’ GTAAGTAAAGTGCCACCAGCC
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Human EGFR forward: 5’GCAGCGATGCGACCCTCCGGG and reverse:
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5’-CTATTCCGTTACACACTTTGCGG
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Human b-actin: forward 5’ CCGACAGGATGCAGAAGGAG and reverse 5’
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CATCTGCTGGAAGGTGGACA
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LC-MS/MS Quantitation of Capmatinib and Crizotinib in Mouse Plasma and Tumor
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For mouse plasma sample analysis, 25 L of each sample was precipitated with 200 L of
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acetonitrile. This suspension was vortexed for 30 min and centrifuged at 4k rpm for 15 min,
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after which 100 L of the extract was aliquoted and mixed with 200 L of acetonitrile/water (1/2,
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v/v) prior to LC-MS/MS analysis. The extracted plasma samples were analyzed on a Waters
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Acquity UPLC system coupled with a Waters Xevo TQ-S triple quadrupole mass spectrometer
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operated at positive mode. The capillary voltage was set to 0.5 kv and collision energy to 32 ev.
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Capmatinib (purchased from Matrix Scientific Products (Columbia, SC)) and crizotinib
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(purchased from LC Laboratories (Woburn, MA)) were separated using a Waters Acquity UPLC
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BEH C18 column (1.7 µm, 2.1 x 30 mm) and detected by a multiple reaction monitoring
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transition, m/z 413.04>354.07 for capmatinib and m/z 450.04>260.18 for crizotinib, respectively.
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The mobile phase A was 0.1% acetic acid/water and B was 0.1% acetic acid/acetonitrile. The
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LC gradient was 10% B (0-0.3 min), 10-95% B (0.3-1.3 min), 95% B (1.3-1.7 min), 10% B (1.7-
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2.0 min) and the flow rate was 0.5 mL/min. The column temperature was 40 C. The injection
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volume was 2 µL. Under these conditions, the retention time was 0.85 min for capmatinib and
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0.74 min for crizotinib. The method was validated with an analytical range of 1 – 1000 ng of
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capmatinib and crizotinib in untreated CD-1 mouse plasma, respectively.
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Mouse tumor tissue samples were homogenized in methanol:water (80:20, v/v) to a
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concentration of 100 mg (tissue)/mL. The homogenates were vortexed for 10 min and
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centrifuged at 15k rpm for 5 min, then 100 L of the supernatant was transferred into an HPLC
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vial for LC-MS/MS analysis. The tissue homogenates were analyzed by using the same method
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as described above. The method was validated with an analytical range of 1 – 1000 ng/mL of
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capmatinib and crizotinib in untreated mouse tumor tissue homogenates, respectively.
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Whole Exome Sequencing
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Library Construction and Sequencing
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The sequencing libraries were prepared using the KAPA library prep protocol (catalog number
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KK8234, KAPA Biosystems, Wilmington, MA). The exomes were captured using the SureSelect
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XT Human All Exon V5 kit (Agilent Technologies, Santa Clara, CA). Samples were then
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sequenced 2x100 bp to about 340x depth on the Illumina HiSeq 2000.
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BAM File Generation
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The raw output (BCL) files of an Illumina sequencer were converted to FASTQ files using
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Illumina's offline basecalling software CASAVA version 1.8.2. The FASTQ files were then
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aligned to the reference genome (hg19 for human) using BWA version 0.7.0 5 for DNA samples
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with parameters suitable for a given aligner. The aligned BAM files are subjected to mark
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duplication, re-alignment, and re-caliberation using Picard version 1.112
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(http://picard.sourceforge.net) and GATK version 1.5 6 when applicable before any downstream
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analysis are conducted.
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Whole Genome Low Pass Sequencing
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Library Construction and Sequencing
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The Illumina compatible libraries were prepared using KAPA DNA Library preparation kit
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(Catalog No. KK8232) as per the manufacturer’s protocol. In brief, DNA was fragmented to a
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median size of 200bp by sonication. Fragmented DNA ends were polished and 5′-
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phosphorylated. After addition of 3′-A to the ends, indexed Y-adapters were ligated and the
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samples were PCR amplified. The resulting DNA libraries were quantified and validated by
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qPCR, and sequenced on Illumina’s HiSeq 2000 in a paired-end read format for 76 cycles. The
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resulting BCL files containing the sequence data were converted into “.fastq.gz” files and
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individual sample libraries were demultiplexed using CASAVA version 1.8.2 with no
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mismatches.
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RNA Sequencing
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Library Construction and Sequencing
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The Illumina compatible libraries were prepared using Illumina’s TruSeq RNA Sample Prep kit
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v2, as per the manufacturer’s protocol. In brief, Poly-A RNA was enriched using Oligo-dT beads.
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Enriched Poly-A RNA was fragmented to a median size of 150bp using chemical fragmentation
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and converted into double stranded cDNA. Ends of the double stranded cDNA were polished,
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5′-phosphorylated, and 3’-A tailed for ligation of the Y-shaped indexed adapters. Adapter ligated
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DNA fragments were PCR amplified, quantified and validated by qPCR, and sequenced on
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Illumina’s HiSeq 2000 in a paired-end read format for 76 cycles. The resulting BCL files
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containing the sequence data were converted into “.fastq.gz” files & individual sample libraries
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were demultiplexed using CASAVA version 1.8.2 with no mismatches.
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BAM File Generation
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RNA sequencing BAM files were generated and analyzed using the Pipeline for RNAseq Data
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Analysis (PRADA) (http://sourceforge.net/projects/prada/) 7. In brief, PRADA uses Burroughs-
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Wheeler alignment, Samtools, and Genome Analysis Toolkit to align RNAseq reads to a
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reference database composed of whole genome sequences (hg19) and transcriptome
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sequences (Ensembl64). Details of the PRADA pipeline are described in its manuscript.
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Targeted Resequencing
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Library Construction and Sequencing
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The Illumina compatible libraries were prepared using KAPA DNA Library preparation kit
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(Catalog No. KK8232) as per the manufacturer’s protocol. In brief, DNA was fragmented to a
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median size of 200bp by sonication. Fragmented DNA ends were polished and 5′-
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phosphorylated. After addition of 3′-A to the ends, indexed Y-adapters were ligated and the
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samples were PCR amplified. The resulting DNA libraries were enriched for targeted regions
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using NimbleGen SeqCap EZ Choice Library 4 RXN (Catalog No. 06740251001) and
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NimbleGen SeqCap EZ Reagent Kit Plus v2 (Catalog No. 06953247001) as per the
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manufacturer’s protocol. The enriched libraries were quantified and validated by qPCR, and
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sequenced on Illumina’s HiSeq 2000 in a paired-end read format for 76 cycles. The resulting
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BCL files containing the sequence data were converted into “.fastq.gz” files and individual
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sample libraries were demultiplexed using CASAVA version 1.8.2 with no mismatches.
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BAM File Generation
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Sequencing FASTQ files were aligned to the reference genome (hg19 for human) and
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processed to BAM files by the same pipeline as in whole exome sequencing.
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Pacific Biosciences (PacBio) Long Read Sequencing
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Library Construction and Sequencing
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The DNA libraries were prepared following the Pacific Biosciences 20 kb Template Preparation
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Using BluePippin Size-Selection System protocol. No DNA shearing was performed since the
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samples were already fragmented. The sheared DNA was size selected on a BluePippin
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system (Sage Science Inc., Beverly, MA, USA) using a cutoff range of 7 kb to 50 kb. The DNA
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Damage repair, End repair and SMRT bell ligation steps were performed as described in the
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template preparation protocol with the SMRTbell Template Prep Kit 1.0 reagents (Pacific
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Biosciences, Menlo Park, CA, USA) . The sequencing primer annealing and the P6 polymerase
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binding reactions were prepared according to the BindingCalculator (Pacific Biosciences
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BindingCalculator-master_v2.3.1.1). The libraries were sequenced on a PacBio RSII instrument
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at a loading concentration (on-plate) of 80pM, 90pM and 100pM using the MagBead
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OneCellPerWell v1 collection protocol, DNA sequencing kit 4.0, SMRT cells v3 and 4 hours
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movies.
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Filtering the sequencing reads
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Reads and subreads were filtered based on their length and quality values, using smrtpipe.py
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from the SMRT-Analysis package.
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Structural Variation Analysis
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Canu (version 1.2) 8 was used for assembling the filtered PacBio sequence subreads with the
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parameters suggested for low coverage data. The assembled contigs were aligned by nucmer
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(version 3.23) 9 to the human genome reference (hg19) and the contigs having sequence
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fragments aligned to the MET-CAPZA2 region of chromosome 7 were selected for structural
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variation analysis. For the selected contigs, we performed a blastn search 10 against mouse
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genome using the sequence fragments aligned to the MET-CAPZA2 region of hg19 in order to
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make sure that they originated from human (Supplementary Table 3). Sequence framents
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shared by two contigs were identified with pairwise alignment of the contigs using the nucmer.
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Two contigs were considered to be connected only if they shared a sequence fragment which
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was at least 5,000 bp long with the minimum 99% identity. The high confident shared sequence
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fragments were used for connecting the contigs into a circular form in the HF3035. In HF3077,
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only two contigs (tig01141776 and tig01141835) were aligned to the MET-CAPZA2 region of
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chromosome 7, and the two contigs shared 621 bp long sequence with 95.6% identity between
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the 3’ end of tig01141776 and the 5’ end of tig01141835.
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Gene Fusion and Gene Expression Analysis
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To detect transcript fusions, PRADA aligned RNAseq reads to a reference database composed
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of whole genome sequences (hg19) and transcriptome sequences (Ensembl64). Two lines of
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evidence were required for identification of a gene fusion: 1) a minimum of two discordant read
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pairs mapping to a candidate gene pair; 2) a minimum of one junction spanning read mapping to
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a junction that connected exons between the candidate gene pair, with its pair mate mapping to
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the either of the two genes. Several filters were applied to remove false positives and artifacts,
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of which the most prominent is based on significant sequence similarity between the two fusion
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genes (using BLASTN, Expect value = 0.01). Gene expression was measured as ‘reads per
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kilobase per million’ (RPKM) to normalize for gene length and library size. Specific details of the
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PRADA pipeline are described elsewhere 7.
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Structural Variant Detection
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To detect structural variants, we applied SpeedSeq 11 (with default parameters) to whole
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genome sequencing from both tumor and matching normal samples. We filtered somatic
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variants by requiring at least 4 reads supporting evidence in a tumor and no reads in its
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matching normal.
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EGFR Intragenic Rearrangement
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General User dEfined Supervised Search for intragenic fusion (GUESS-if), a module of PRADA,
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was also used to search for EGFR intragenic rearrangements, as previously described 12. In
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brief, using the same rationale as in PRADA gene fusion identification, GUESS-if looked for
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spanning reads for abnormal junctions that were not present in known transcripts. To assure a
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high accuracy, we required at least 10 reads spanning exon 1-8 of EGFR.
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Validation of Somatic Single Nucleotide Variants
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To validate our somatic single nucleotide mutation calls, we performed targeted resequencing at
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high coverage (>1,400x). We selected 792 unique bases, which had been found to be mutated
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in tumor, neurosphere, or xenografts but not in all of them. These sites corresponded to
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1368sSNVs. In total, 1287of 1368mutations called from the exome sequencing data were
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detected in the high coverage data, resulting in a true positive validation rate of 94%. Evidence
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for recovered somatic mutation was observed in 1001 of 2646 wild type nucleotides. The variant
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allelic fractions (VAFs), i.e. the number of reads harboring the variant allele divided by all reads
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covering to that base, of exome and validation sequencing were highly correlated (Pearson
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correlation = 0.92).
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Somatic Single Nucleotide Variant Calling
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Somatic single nucleotide variants (sSNVs) from tumor and patient-matched normal samples
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were detected by using MuTect algorithm (version 1.14) with default parameters 13. The search
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for somatic small insertion/deletions (Indels) was performed by using Pindel 14 with tumor and
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patient-matched normal samples. All sSNVs and small indels were annotated by ANNOVAR
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(version 2012-10-23) 15. Only exonic or splicing sSNVs were selected for analysis. Mutation
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counts for individual samples are available in Supplementary Table 4.
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Inference of Cellular Frequency and Mutational Clusters
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We defined cellular frequency of a mutation as the fraction of cells harboring the mutation.
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Estimation of cellular frequency was performed using PyClone version 0.12.7
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. For each set of
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patient, neurosphere, and xenograft samples, PyClone was run on the somatic mutations whose
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sites were covered over all the samples using multi-sample joint analysis mode with PyClone
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beta binomial density and parental copy number priors. Allelic copy numbers were estimated by
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applying Sequenza 17 to exome sequencing data. Default options for PyClone were used. To
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avoid potential artifacts from sequencing coverage, we limited the analysis to the mutations at
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the sites covered with at least 50X over all samples from a same patient. PyClone inferred
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clusters of mutations whose cellular frequencies co-vary over samples. Our analysis was limited
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only to mutation clusters with at least two mutations.
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Removing Putative Mouse Reads in Short Read Sequencing Data
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Sequencing reads derived from xenograft samples are a mixture of reads from human and
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mouse. We utilized Xenome 18 to select sequencing reads arising from human. Then, the
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selected human reads selected were aligned to the human genome using the same pipeline as
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in patient and neurosphere samples.
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Identification of Copy Numbers from Low Pass Sequence Data
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We used NBICSeq version 0.5.2 19 with bin size 1000 bps and BIC penalty 3 to estimate
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somatic copy number alterations in low pass sequencing data from tumor and patient-matched
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normal samples.
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Detecting TERT Promoter Mutations
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We evaluated whole genome low pass sequencing and whole exome sequencing for the
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presence of TERT mutations in a supervised way using GATK pileup. We required minimum 2
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variant alleles (combined from WGS and WES) for detection of TERT promoter mutations.
Variant change
Variant site
Patients
C228T
5:1295228-1295228
7
C250T
5:1295250-1295250
5
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Detecting ATRX Indels
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Indels were called using Pindel (Version 0.2.4t) with the default parameters except maximum
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allowed mismatch rate being 0.1 14. Somatic indels were further filtered to require a minimum 5
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supporting tumor reads.
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Analysis of B-allele-frequency segments
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B-allele-frequency segments were inferred by applying Sequenza (Version 2.1.1)
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exome sequencing data with the default parameters. Analysis of B-allele fractions using whole
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genome sequencing in our sample cohort revealed loss of heterozygosity (LOH) of chromosome
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10 in two cases with diploid chromosome 10, suggesting these cases had first lost a single copy
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of the chromosome which was subsequently duplicated (Supplementary Fig. 1). We evaluated
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chromosome 10 LOH using Affymetrix SNP6 profiles from 320 IDH-wildtype TCGA glioblastoma
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underscoring the importance of aberrations in chromosome 10 in gliomagenesis and evolution
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(Supplementary Fig. 6).
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Data used for longitudinal analysis in glioma patient tumors
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Segmented copy number profiles for thirteen TCGA GBM patients and fourteen TCGA LGG
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patients were were obtained from the TCGA portal https://tcga-data.nci.nih.gov/tcga/. Copy
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number profiles for ten patients from MD Anderson Cancer Center (MDACC) and fourteen
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patients from either Samsung Medical Center (SMC) or Seoul National University Hospital
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(SNUH) were previously processed 20,21. Additional copy number data for seven patients from
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MD Anderson were generated by applying NBICseq version 0.5.219 to low pass whole genome
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sequencing. For fusion detection and structural variant calling, the same pipelines as described
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in the corresponding method subsections were applied for unaligned RNA sequencing files and
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whole genome sequencing BAM files from TCGA GBM, TCGA LGG, and MD Anderson
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patients. Sequencing data for the TCGA cohort were downloaded from CGHub. Fusion calls for
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Samsung Medical Center cohort patients were previously processed 21. Shown below is a
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summary table of data used in the analysis.
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to whole
, and found that 27 of 52 tumors with diploid chromosome 10 similarly showed LOH,
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Table: The number of patients used in the longitudinal analysis
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Cohort
CNV
RNASEQ
WGS
MDACC
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9
7
SMC/SNUH
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0
0
TCGA GBM
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6
10
TCGA LGG
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14
13
Total
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Note: Patients do not necessarily have both RNAseq and WGS.
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Predicting double minute candidates
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After visualizing segmented copy numbers in the Integrative Genomics Viewer (IGV) 22, we
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manually scrutinized potential extrachromosomal DNA candidate regions by searching for
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complex patterns of copy number amplifications. In cases where structural variations and gene
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fusions were available, we projected those variation breakpoints onto the copy number IGV view
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plots to get additional evidence on presence of potential extrachromosomal DNAs.
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Statistical Analysis
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We conducted all computations with R 3.0.13 and used standard statistical tests as appropriate
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More explanation on CAPZA2-MET fusion transcripts
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Chimeric RNA fusions have been previously reported in GBM
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therapeutically targetable, in particular when involving receptor tyrosine kinases
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We performed RNA sequencing and detected fusion transcripts in all samples except
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for a single neurosphere line (HF3203) with disqualifying quality control values 7. From
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this unbiased screen, multiple fusions joining the CAPZA2 coding start with the 5’ UTR
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of MET were identified in the primary tumors of HF3035, HF3077 and HF3055 (Fig. 3b).
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Additional CAPZA2-MET variants resulted in an in-frame transcript consisting of
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CAPZA2 exon 1 and MET starting from exon 3 (HF3035, HF3077) and exon 6
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and may be
26,27.
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(HF3035). The CAPZA2-MET fusions associated with outlier gene expression of MET
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while CAPZA2 expression was comparable between samples with and without
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CAPZA2-MET fusions (Supplementary Fig. 7a). The presence of multiple parallel fusion
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transcripts suggested complex chromosomal rearrangements, which associated with
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focal amplification of a 200 kb area on 7q31 (Fig. 3b). Amplification of the 7q31 genomic
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area carrying the adjacent CAPZA2 and MET genes has been previously reported in
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glioma
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glioblastoma we analyzed the DNA copy number profiles of 486 TCGA IDH wildtype
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glioblastoma samples. A focal amplification of the MET locus ranging in size from 150kb
368
to 5.1 Mb which associated with a highly significant increase in expression relative to
369
samples with broad 7q amplification or diploid MET copy number was identified in ten
370
cases (2.1%) (Supplementary Fig. 7b). RNAsequencing data was available for one of
371
the ten TCGA cases and no fusions involving MET were detected in those samples.
372
CAPZA2-MET fusions have been infrequently reported in other cancers
373
response of a glioblastoma carrying MET amplification to MET and ALK inhibiting agent
374
crizotinib has been recorded 31.
28.
To assess the frequency of MET-activating somatic alterations in
29,30.
Clinical
375
In spite of convincing evidence supporting fusion events in the GBM samples
376
from HF3035, HF3055 and HF3077, no sequencing reads manifesting the presence of
377
CAPZA2-MET fusion transcripts or the focal 7q31 genomic amplification were identified
378
in the HF3055 and HF3077 neurospheres and only weak support was found in the
379
HF3035 neurosphere. However, identical CAPZA2-MET fusions and 7q31 DNA
380
amplifications resurfaced at high frequency in all xenografts derived from the HF3035
381
and HF3077 neurospheres, with identical breakpoints (Supplementary Fig. 3b). None of
382
the HF3055 xenografts carried CAPZA2-MET fusions or 7q31 amplification, in line with
383
the absence of focal 7q31 amplification in the primary HF3055 tumor. To exclude the
384
possibility that the CAPZA2-MET fusion events were artifacts resulting from sequencing
385
we validated the event in all samples from HF3035 using RT-PCR, which confirmed
386
both wildtype MET and CAPZA2-MET mRNA in the tumor and PDX but not
387
neurosphere (Supplementary Fig. 3a). MET protein was abundantly present in the
388
HF3035 and HF3077 tumors as measured using immunohistochemistry, undetectable in
389
the neurospheres and re-expressed in the PDX (Supplementary Fig. 3c).
13
390
14
391
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