To confirm large rearrangements found by MLPA, we used the chromosomal microarray technique CytoScan HD (Affymetrix), according to the manufacturer's protocol. The high‐density, whole‐genome CytoScan Array includes 2.69 million markers for copy number analysis, which are representative of DNA sequences distributed throughout the genome. Chromosome Analysis Suite software (ChAS software 3.1) was used to analyze and visualize microarray data. Deletions and duplications with more than 50 markers involved and size of 250 bp or more were considered in ChAS analysis. The following parameters were also considered: mean marker distance, copy number state, smooth signal and log2 ratio. Data were compared with the Database of Genomic Variants (DGV‐http://dgv.tcag.ca/dgv/app/home ), affymetrix DGV (a database created by affymetrix that includes deletions and duplications found by CytoScan HD) and common artifacts database (a database created by affymetrix that includes artifacts commonly found by CytoScan HD, used to exclude this type of alteration).
Cytoscan hd
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom
The CytoScan HD is a high-density microarray platform designed for cytogenetic research and clinical applications. It provides comprehensive coverage of the human genome, enabling the detection of copy number variations and chromosomal abnormalities.
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Lab products found in correlation
Chromosome analysis suite, by Thermo Fisher Scientific (6 mentions)
Chromosome analysis suite software, by Thermo Fisher Scientific (5 mentions)
Genechip scanner 3000 7g, by Thermo Fisher Scientific (4 mentions)
Qiaamp dna mini kit, by Qiagen (4 mentions)
Chromosome analysis suite chas software, by Thermo Fisher Scientific (3 mentions)
Genechip 250k nsp, by Thermo Fisher Scientific (3 mentions)
Chromosome analysis suite chas, by Thermo Fisher Scientific (3 mentions)
Genotyping console, by Thermo Fisher Scientific (3 mentions)
Cytoscan hd array, by Thermo Fisher Scientific (3 mentions)
Chromosome analysis suite 2, by Thermo Fisher Scientific (3 mentions)
Oncoscan cnv plus, by Thermo Fisher Scientific (2 mentions)
Oncoscan, by Thermo Fisher Scientific (2 mentions)
Cytoscan 750k, by Thermo Fisher Scientific (2 mentions)
Novaseq 6000, by Illumina (2 mentions)
Quantitect sybr green pcr kit, by Qiagen (2 mentions)
Cytoscan hd, by BioDiscovery (2 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Taqman copy number assay, by Thermo Fisher Scientific (1 mentions)
Taqman rnasep control reagent, by Thermo Fisher Scientific (1 mentions)
Dneasy blood and tissue kit, by Qiagen (1 mentions)
101 protocols using cytoscan hd
1
Confirming Genetic Rearrangements with Microarray
2
Comprehensive Genomic Profiling of GIST
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Whole chromosome gains and losses and copy number aberrations (deletions and duplications) were determined in quadruple WT cases using CytoScan HD or Oncoscan CNV Plus array (Thermo Fisher Scientific, Milan, IT) for FF or FFPE specimens, respectively. Copy number data were analyzed and visualized with Chas 3.1 (Thermo Fisher Scientific).
As reference control dataset we used copy number data available online from other GIST samples: GSE93077 from Schaefer et al14 and GSE20709 from Astolfi et al15 GSE93077 is a series of 9 KIT mutated cases for which copy number data produced using CytoScan HD Array was available. GSE20709 is a series of 21 KIT/PDGFRA and 4 SDHx mutated GIST analyzed with SNP6.0 array. Moreover, SNP6.0 array data of additional 10 KIT/PDGFRA mutated GIST were used. Global copy number alteration were analyzed with Chas 3.1 and FGF3/FGF4 locus was manually checked for putative focal alterations.
As reference control dataset we used copy number data available online from other GIST samples: GSE93077 from Schaefer et al
3
Comprehensive Cytogenomic Profiling for B-ALL
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The City of Hope Cytogenetics Laboratory is certified under the CLIA 88 as qualified to perform high-complexity clinical laboratory testing. Testing includes G-banded chromosome analysis, FISH, and whole-genome oncology microarray (SNP array) for hematological malignancies (CytoScan HD). The laboratory is approved by the Children’s Oncology Group (COG) to perform testing for acute leukemia.
We performed Chromosomal Microarray Analysis (CMA) using the Affymetrix CytoScan HD platform (Thermo Fisher Scientific). This microarray consists of 2 696 550 oligonucleotide probes across the genome including 1 953 246 unique nonpolymorphic probes and 743 304 SNP probes. Patient hybridization parameters are compared with data derived from phenotypically normal individuals. We applied this microarray and associated software (chromosome analysis suite) to identify DNA copy number alterations and extended regions of copy neutral loss of heterozygosity, known in B-lymphoblastic leukemia.
Final results included pathogenic, likely pathogenic, large regions of copy neutral loss/absence of heterozygosity, and variants of uncertain significance. FISH panel for standard-risk ALL was performed on all cases, including BCR-ABL1-ASS1 with 1% background cutoff, IGH with 2% background cutoff, +4/+10 with 2% background cutoff, ETV6-RUNX1 with 2% background cutoff, and KMT2A with 3% background cutoff.
We performed Chromosomal Microarray Analysis (CMA) using the Affymetrix CytoScan HD platform (Thermo Fisher Scientific). This microarray consists of 2 696 550 oligonucleotide probes across the genome including 1 953 246 unique nonpolymorphic probes and 743 304 SNP probes. Patient hybridization parameters are compared with data derived from phenotypically normal individuals. We applied this microarray and associated software (chromosome analysis suite) to identify DNA copy number alterations and extended regions of copy neutral loss of heterozygosity, known in B-lymphoblastic leukemia.
Final results included pathogenic, likely pathogenic, large regions of copy neutral loss/absence of heterozygosity, and variants of uncertain significance. FISH panel for standard-risk ALL was performed on all cases, including BCR-ABL1-ASS1 with 1% background cutoff, IGH with 2% background cutoff, +4/+10 with 2% background cutoff, ETV6-RUNX1 with 2% background cutoff, and KMT2A with 3% background cutoff.
4
Comprehensive Genome-wide CNV and BAF Analysis
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CNV and BAF analyses were performed using CytoScan HD (ThermoFisher Scientific) harboring more than 2.4 million CNV markers and approximately 750,000 SNP markers. The log ratio and B-allele frequency were calculated using Rawcopy R package [26 (link)]. We selected CNVs larger than 100-kb, using log-ratio thresholds of > +0.2 for gains and < −0.3 for deletions, as suggested previously [26 (link)], and these CNVs were annotated using AnnotSV (Annotation and Ranking of Human Structural Variations) (https://www.lbgi.fr/AnnotSV/runjob ). We searched for known pathogenic CNVs and CNVs of unknown significance absent from multiple public databases adopted in AnnotSV. CNVs located on segmental duplications were regarded as begin CNVs. Violin plot for BAFs was made with snp.profile.Rdata by ggplot2 (https://ggplot2.tidyverse.org/reference/index.html ).
5
Cytoscan HD SNP Array Analysis
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SNPa (Cytoscan HD, Thermo Fisher Scientific) data were generated during clinical molecular diagnostics from the corresponding primary tumor or, if available, the bone marrow sample that was processed for scRNA-seq. Metastatic tumor cells in the bone marrow were enriched by MACS sorting for GD2 as described previously4 (link). DNA was extracted from purified bone marrow metastatic NB cells or fresh frozen tumor pieces and 50–200 ng DNA was used for SNPa analysis as described before4 (link). Data analysis and CEL files were processed using ChAS 4.3 (Thermo Fisher Scientific). Copy number (log2 ratio) and bi-allele frequency tracks were visualized and predicted genomic segments (gains, losses, copy number) were manually curated using the IGV browser and plug-in VARAN-GIE69 (link). TDF files were converted to BEDGRAPH format using IGV v2.9.4, and the logrr values in the latter files were directly plotted.
6
Metastatic Neuroblastoma SNP Array Analysis
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High‐resolution SNP arrays analysis was performed in 41 metastatic NB patients. Out of the 41, 29 were classified as stage M and 12 as stage MS (Metastatic Special). We included MS cases to identify SCA differences between both metastatic subgroups. Whole‐genome copy number variations were analyzed by SNP arrays (CytoScan HD, Thermo Fisher Scientific, Inc., Waltham, MA, USA) as previously described [9 ]. Briefly, isolated DNA from fresh tumor was fragmented by Nsp I digestion and further ligated to adaptor followed by PCR amplification. The PCR product was hybridized using Affymetrix CytoScan HD Array Gene Chip and processed with the Fluidic Station ( Thermo Fisher Scientific, Inc.). SNP array results were analyzed with Chromosome Analysis Suite software (chas v3.1 ; Thermo Fisher Scientific, Inc.). The annotation version used by the chas software is based on the February 2009 human reference sequence GRCh37 (hg19).
7
Validating MYCN Copy Number Estimation
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For validation of WES-based estimation of MYCN copy numbers, a SNP array (Cytoscan HD, ThermoFisher Scientific, #901835) was performed for selected patient samples as in ref. 15 (link). CEL files with intensity probe signals were analysed using the Chromosome Analysis Suite (ChAS) software version 4.1.0.90 (r29400) and converted to CYCHP files. Copy numbers are visualised using the allele difference plot, weighted log2 ratio and the smoothed signal. The smoothed signal is used to directly estimate copy numbers.
MYCN copy number was detected on FFPE tissue on single-cell level using FISH analysis. The hybridisation probe XL MYCN amp (MetaSystems) consists of a green-labelled probe hybridising to the MYCN gene region at 2p24 and an orange-labelled probe hybridising to the NMI gene region at 2q23 as reference. Data interpretation was done according to INRG Biology guidelines69 (link).
MYCN copy number was detected on FFPE tissue on single-cell level using FISH analysis. The hybridisation probe XL MYCN amp (MetaSystems) consists of a green-labelled probe hybridising to the MYCN gene region at 2p24 and an orange-labelled probe hybridising to the NMI gene region at 2q23 as reference. Data interpretation was done according to INRG Biology guidelines69 (link).
8
CytoScan HD Assay for CMA
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The genomic DNA (gDNA) for CMA was analyzed using the CytoScan HD assay following the manufacturer’s protocol (ThermoFisher Scientific, USA). The data were analyzed using the Chromosome Analysis Suite (ChAs 3.0) software, where the signal for the sample was compared with a reference set. Differences in signal between the sample and reference were expressed as a log2 ratio and represent relative intensity for each marker. A discrete copy number value was determined from the relative intensity data. CNVs were confirmed by quantitative polymerase chain reaction technology.
9
Chromosomal Microarrays for Genetic Analysis
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Chromosomal microarrays were assessed in DNA from PBMCs (n = 12) or PB CD19+ purified cells (n = 28). DNA was amplified, labeled, and hybridized to CytoScan HD (n = 30) or CytoScan 750K (n = 10) platforms (ThermoFisher Scientific, Eugene, OR, USA) according to manufacturer’s protocols. Copy number variants found as benign polymorphisms in the Database of Genomic Variants (http://dgv.tcag.ca/dgv/app/home ; accessed on 4 April 2022) were excluded. The remaining aberrations, irrespective of size, were collected and reported using annotations of genome version GRCh37/hg19. CNV counting for risk classification of patients was performed using previously described criteria [27 (link),28 (link)]. As the current analysis pipelines used for OGM results do not enable copy-number neutral loss of heterozygosity (CN-LOH) detection, these abnormalities were not considered in the present study.
10
Copy Number Assessment of FGF Insulator
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Copy number assessments were performed using TaqMan copy number assays (Thermo Fisher Scientific). Evaluation of the FGF insulator copy number was performed using Hs06280902_cn and Hs06332446_cn FAM‐labelled probes, covering the upstream and downstream regions of the insulator, respectively. The FGF4 copy number was evaluated as previously described7 (link). Hs03800758_cn and Hs03782780_cn FAM‐labelled probes targeting ENDOD1 and XXRA1 located outside FGF4 duplication margins, respectively, were used for copy number normalization. TaqMan RNaseP Control Reagent (VIC‐labelled; Thermo Fisher Scientific) was used as an internal reference control. The copy number was estimated using the DDCt method and using a normal diploid sample as the calibrator. Moreover, high-density copy number array data (CytoScan HD or Oncoscan CNV Plus, Thermo Fisher Scientific) of quadruple WT GISTs were obtained from our previously published paper7 (link).
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