Raw MIP data from three additional primary-metastatic ES pairs were obtained from the Huntsman Cancer Institute, Salt Lake City, Utah (42 (link)). The original source material was clinically-archived, formalin-fixed paraffin-embedded (FFPE) scrolls that were retrieved from 3 individual patients diagnosed with ES. Primary tumor samples were from diagnostic biopsies prior to chemotherapy. The raw MIP data from the completed assay was loaded into Nexus Copy Number (BioDiscovery, Inc., El Segundo, CA) for copy number detection using default settings.
Nexus copy number
Manufactured by BioDiscovery
Sourced in United States
Nexus Copy Number is a laboratory equipment that measures and analyzes copy number variations in DNA samples. It provides quantitative data on the number of copies of specific genomic regions.
Automatically generated - may contain errors
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18 protocols using nexus copy number
1
Copy Number Detection of Primary-Metastatic ES Pairs
2
Whole Genome Sequencing and CNV Analysis
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Genomic DNA extracted from blood was used to perform WGS for Patient 1 via the commercial provider Macrogen (South Korea) using Illumina HiSeqX technology. Sequencing was performed to an average sequence depth of 28.5×. The resulting sequence files were aligned to hg38 using Isaac Aligner [20 (link)]. Subsequently, WGS data were analyzed for CNVs using the software Nexus Copy Number (BioDiscovery, El Segundo, CA, USA).
3
Genomic DNA Amplification and Labeling
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Whole-genome amplification (WGA) of the DNA samples (approximately 10 ng) was done using the REPLI-g Mini Kit (Qiagen Corp), and the amplified DNA was verified using a Nanodrop instrument (Thermo Scientific, Waltham, MA, USA) or a 3% agarose gel to determine the quality and quantity of the product. Amplified DNA (2.5 μg) and sex-matched control DNA (Promega, Madison, WI, USA) were digested using AluI (Promega) and RsaI (Promega) for 2 hours and then purified using a QIAprep Spin Miniprep Kit (Qiagen Corp) before being labeled for 2 hours with Cyanine (Cy) 3 dye-labeled deoxyuridine triphosphate (Cy3-dUTP) and Cy5-dUTP (Promega), respectively, in a random priming reaction using Bioprime Array CGH Genomic Labeling Module (Life Technologies, Carlsbad, CA, USA). Unincorporated nucleotides were removed using Microcon YM-30 columns (Millipore, Bedford, MA, USA) before both Cy3- and Cy5-labeled DNA were combined. Human oligonucleotide-based microarrays (4 × 44 K, Agilent Technologies, Santa Clara, CA, USA) were hybridized for 40 hours at 65°C, washed with the manufacturer’s recommended solutions, and scanned (G2565BA, Agilent Technologies). The resulting file was then extracted using the Agilent Feature Extraction™ software (Agilent Technologies) before analysis using the Nexus Copy Number (BioDiscovery, Hawthorne, CA, USA).
4
DNA Copy Number Profiling by WES
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DNA copy number was determined from WES read count data using the BAM MultiScale Reference (MSR) Algorithm within Nexus Copy Number (Biodiscovery, El Segundo CA). First, realigned bam files for the non-neoplastic control samples were pooled to create a common reference file using the Multiscale BAM Reference Builder module. Realigned bam files for each of the test DNA samples were then processed relative to the common reference file, using the MSR algorithm to generate pseudo-log ratios based on read depth.
Discrete regions of DNA copy number imbalance were defined using the FASST2 Segmentation algorithm within Nexus Copy Number, using default parameters. Briefly, the significance threshold for segmentation was set at 1x10-6, with a minimum of three probes per segment and a maximum spacing of 1Mb between adjacent probes before breaking a segment. The ratio thresholds for single copy gain and single copy loss were set at 0.18 and -0.18, respectively, and thresholds for high amplitude gains and losses were set at 0.6 and -1.0 respectively. Data analysis was restricted to autosomes due to the inclusion of dogs of both sexes in the study cohort.
Discrete regions of DNA copy number imbalance were defined using the FASST2 Segmentation algorithm within Nexus Copy Number, using default parameters. Briefly, the significance threshold for segmentation was set at 1x10-6, with a minimum of three probes per segment and a maximum spacing of 1Mb between adjacent probes before breaking a segment. The ratio thresholds for single copy gain and single copy loss were set at 0.18 and -0.18, respectively, and thresholds for high amplitude gains and losses were set at 0.6 and -1.0 respectively. Data analysis was restricted to autosomes due to the inclusion of dogs of both sexes in the study cohort.
5
aCGH Analysis of CEBPD and MYC in UBUC
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Reanalysis of our published aCGH dataset containing 40 UBUC samples10 was performed by using Nexus Copy Number software (BioDiscovery, USA) as previously described23 to profile the status of CEBPD and MYC coamplification. For this analysis, the copy‐number gain was defined as log2 ratio >0.2.
6
Genome-wide aCGH for CNV Detection
7
Genome-Wide Copy Number Variation Analysis
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Agilent 8X60K CGH microarrays (Agilent Technologies, Inc., Santa Clara, CA) were performed to determine DNA copy number change of all patient samples following the manufacturer's protocol. Slides were scanned on an Agilent DNA Microarray Scanner, using standard settings. Microarray images were processed with Agilent Feature Extraction software to generate normalized, background-subtracted feature intensities. The microarray data were further analyzed with Nexus Copy number (BioDiscovery, Inc., Hawthorne, CA). In order to determine copy number variations (CNV), a log2 ratio threshold was set as (0.585/−0.8) for ‘gain/loss calls’. Aberrant segments from all samples with frequency higher than 25% were selected.
8
Profiling Tumor and Normal DNA by WES
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Tumor and normal DNA samples were profiled by WES in five batches using SureSelect Exome V5 or V6 + UTR library capture (Agilent, USA) and sequenced on Illumina HiSeq 2500 or HiSeq 4000 (see Supplemental Methods for quality‐control metrics). Duplicate reads and PCR artifacts were removed using Picard Tool function MarkDuplicates before import into Nexus Copy Number™ (version 8.0, BioDiscovery, CA, USA) for CNA analysis. For each tumor sample, the matched normal DNA from blood of the same patient was used in the ngCGH (matched) processing according to the software instructions. For two liver metastatic and four primary tumor samples, normal matched DNA was not available, and they were processed using a pooled reference that was built with the normal DNA samples of the remaining patients using the BAM Reference Builder utility and according to the recommendations of the manufacturer (BioDiscovery Inc, CA, USA). SNP‐FASST2 Segmentation Algorithm was applied using defaults settings (see Supplemental Methods for settings and quality‐control metrics). Sex chromosomes were excluded from all analyses.
9
SNP Array Analysis and Trio Genotyping
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For patients A and B, SNP array analysis was performed with Affymetrix SNP6.0 arrays according to the manufacturer’s protocol (Santa Clara, CA, USA) as described [6 (link)]. (Affymetrix is now part of ThermoFisher Scientific, Waltham, MA, USA). DNA trio analysis of patient C and parents was done on the 300 K Illumina (San Diego, CA, USA) HumanCytoSNP-12 BeadChip platform [7 (link), 8 (link)] and trioanalyses of patients D and E and their parents were done on the Illumina InfiniumCytoSNP-850K BeadChip platform [8 (link), 9 (link)]. These platforms had (consecutively) become part of our standard clinical laboratory procedure. A cascade of algorithms was applied for copy number analysis and genotyping, including Genome Studio (Illumina), GTC (Affymetrix) and Nexus CopyNumber™ (Biodiscovery, El Segundo, CA, USA). Analysis of LOH (Loss of Heterozygosity) was based on B-allele frequency calculation (BAF), the B-allele representing the minor non-reference allele. Expected values for BAF are 0 for AA, 0.5 for AB and 1 for BB, meaning ∆BAF represents estimated deviation from the expected AB value.
10
CytoScan 750K Array Analysis
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The DNA from MDS was prepared for hybridization to the Affymetrix CytoScan 750 K array (750,000 probes) according to the manufacturer's protocol. A total of 250 ng of isolated DNA per sample was digested with NspI, and the sample was subsequently ligated, PCR-amplified and purified, fragmented, biotin-labelled and hybridized for use in a CytoScan 750 K array (Affymetrix). The data were analysed using the Nexus Copy Number (version 7.5; Biodiscovery Inc.) software programme, and they were normalized using the SNP-FASST2 segmentation algorithm. The normalized probe intensity and allele ratio data were visualized in Nexus v7.5. In addition to the microarray analysis, the TaqMan Copy Number Assay was also used to quantitatively analyse the CN of ROBO1 and ROBO2. The primers and probes were purchased from Applied Biosystems Inc, and the assay was performed according to the manufacturer's instructions. Each replicate was normalized to RPP14 (a reference gene) to obtain a ΔCt (FAM dye Ct- VIC dye Ct), and an average ΔCt for each sample was calculated. All of the samples were normalized to a calibrator sample to determine the ΔΔCt. The relative quantity was 2-ΔΔCt, and the copy number was 2 × relative quantity.
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