OncoScan™ CNV Assay for Research - FAQs

Yes, OSCHP files from both OncoScan CNV Assay (Cat. No. 902293) and OncoScan CNV Plus Assay (Cat. No. 902695) can be opened and viewed simultaneously in Chromosome Analysis Suite (ChAS) Software.
Note: The OncoScan CNV Assay does not contain the somatic mutation probes that are in the OncoScan CNV Plus Assay.

The main difference between the two products is in the reagent kit. The OncoScan CNV Plus Assay contains both somatic mutation probes and copy number probes. The OncoScan CNV Assay contains only copy number probes.

- The OncoScan CNV Plus Reagent Kit contains the OncoScan CNV Plus Somatic Mutation Probe Mix 1.0 (Part No. 902247).
- The OncoScan CNV Reagent Kit does not contain the Somatic Mutation Probe Mix. Instead, it contains Buffer C (Part No. 902696).

The OncoScan CNV Plus Assay detects genome-wide copy number changes, loss of heterozygosity (LOH), and a panel of somatic mutations. The somatic mutation panel includes 74 mutations found in BRAF, KRAS, EGFR, IDH1, IDH2, PTEN, PIK3CA, NRAS, and TP53.

The OncoScan CNV Assay detects genome-wide copy number changes and loss of heterozygosity (LOH).

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Oncoscan CNV Assay is 8 micron, 100 format. 160 µL Hyb mix is added to the array. There should be no air bubble after the hyb mix is added and the chamber should be filled completely.

There is no available equivalent to the "Offset Flag" in the QC and Sample Info tab in ChAS. The "Offset Flag" is a QC metric that is found in the Analysis Dashboard for OncoScan products. This QC metric lets us know if the TuScan algorithm detected that the usual normalization method was off, and therefore required readjustment.

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Tough-Spots® labels are small adhesive stickers used to temporarily seal the backs of cartridge arrays during the overnight hybridization step. They are required to prevent loss of volume due to evaporation through the septa. We recommend using Tough-Spots® labels on Rolls from USA Scientific (Item No. 9185-0000)

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Proper hybridization volume is critical to obtaining an even signal across a given array. Too little volume can lead to black circles in the middle of the array. Too much volume can leak out of the back of the array. The correct hybridization volume leaves enough room for a small air bubble to circulate around the array surface during the overnight hybridization. Here are the recommended hybridization and fill volumes based on the array format:
Array Format; Hybridization Volume; Fill Volume
- 49 Format (Standard); 200 µL; 250 µL
- 64 Format; 200 µL; 250 µL
- 100 Format (Midi); 130 µL; 160 µL
- 169 Format (Mini); 80 µL; 100 µL
- 400 Format (Micro); 80 µL; 100µL

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Key opinion leaders in the Oncology field as well as papers were consulted when choosing the 900 cancer-related genes for the array.

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A minimum of 80ng of DNA is needed per sample for either the OncoScan FFPE or the OncoScan CNV assays.

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The OncoScan assay is enabled by the MIP (molecular inversion probe) assay. The MIP technology was an assay desgined to differentiate between the A/T and G/C channel. Two chips are used to allow differentiation between the channels as the detection is single-color.

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Sample concentration for the OncoScan assay must be performed using a dsDNA specific quantitation method. We strongly recommend using either Quant-it PicoGreen Assay or Qubit dsDNA quantitation kit. Sample concentration determined by UV absorbance or NanoDrop must NOT be used at all in this assay.

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There are no officially recommended stopping points in the OncoScan Assay. For additional information about specific parts of the assay, please contact support.

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These data files are produced that are key to the process:

ARR file - This file includes sample information.
AUDIT file - This file is a log of the sample history.
DAT file - This file is the raw data from the scanner.
CEL file - This file is the gridded and processed data.
xxCHP file - This file is the output of ChAS 3.1 and contains all of the analysis data.
CHPCAR file - This file stores user-annotated calls, interpretations, and modifications made to CHP file segment data (ChAS v2.0 and higher).

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OncoScanGeneBoundaries.r1 lists the approximately 900 OncoScan cancer genes and includes greater than 10 kb on each side of the gene. OncoScanCancerGeneOnly.r1 lists the same approximately 900 OncoScan cancer genes using the start and stop positions of the gene (no additional 10 kb on each side of the gene).

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The algorithm identifies normal diploid markers in the cancer samples. This is particularly important in highly aberrant samples. The normal diploid markers are used to calibrate the signals so that a log2 ratio of 0 (e.g., copy number 2) is achieved. In about 2% of samples, the algorithm cannot identify a sufficient number of “normal diploid” markers, and no normal diploid calibration occurs. This event triggers “low diploid flag = YES.” In this case, the user needs to carefully examine the log2 ratios and verify that re-centering is necessary.

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Celpaircheckstatus checks if the results from the AT channel are consistent with the results from the GC channel and represent the results of the respective channel. This metric will be “out of bounds,” for example, when the AT and GC channels were mispaired between samples or due to sample contamination. We recommend troubleshooting as described in the OncoScan Console User Manual.

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The ChAS software includes a table with the following calls made for each mutation: “high confidence,”, “lower confidence,” and “undetected.” The thresholds for the “high-confidence” calls were established based on separations between the normal reference and mutant calls that resulted in 95% sensitivity and 99% specificity (for lower confidence) and 95% sensitivity and 99.9% specificity (for high confidence) in our spike in experiments. The software provides for visualization of mutant probes versus reference probes representing the wild type to enable users to visually assess this separation for their sample. There is no overlay with copy number data (other than through table summaries).

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Ploidy refers to the baseline copy number observed in the tumor portion of the sample. Ploidy cannot always be estimated. For example, a sample with four copies of each chromosome and no other aberrations looks the same as a sample with two copies of each chromosome.

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This is possible when:

The tumor is nearly homogeneous (i.e., it has a major dominant clone), and the percentage of aberrant cell fraction is 40% or higher.

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This represents the percentage of aberrant cells in a sample:

When reported as “N/A,” it means that the percentage of aberrant cells could not be estimated.
When reported as a percentage (e.g., 60%), it means that 60% of the cells were aberrant.

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Log2 and B-allele frequency.

Linear copy number calls when you mouse over aberrations. In some cases, linear calls are calls in aberrant cell fraction only. In the software, when the percentage of aberrant cells has a percentage value, the linear call for aberrations in that sample is the call in the aberrant cells only. Values will be integers. When the percentage of aberrant cells is “N/A,” the linear call is an average call across the aberrant and non-aberrant cells, and values will be non-integers. You can estimate the copy number in aberrant cells if you have an independent tumor burden estimate.

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We recommend the TuScan algorithm. This algorithm has fixed settings based on 19 probes/call to ensure high sensitivity and specificity at the stated resolution claims. Nexus Express Software for OncoScan FFPE Assay Kit has an additional algorithm for copy number calls from OncoScan FFPE Assay Kit: SNPFASST2 algorithm, developed and supported by BioDiscovery. This allows the user to adjust settings and make custom calls (e.g., with fewer probes). Use cases are:
When breakpoints are clear with fewer probes
When probe re-centering is needed

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Yes, if using the Affymetrix defined workflow and depending on the size of the exon (our algorithm requires 20 contiguous markers to make a call). If using an “open” workflow, the user can define the number of probes they need to claim exon resolution.

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The HapMap concordance of genotyping calls is greater than 99%.

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OncoScan CNV & Oncoscan CNV Plus Assay Kit can detect copy number changes up to 50 copies. As the number of copies increases, the user should bin the calls. For example, copy numbers of 20-50 should be considered as one bin. The recommended bins are: 0, 1, 2, 3, 4, 5-6, 7-10, 11-20, 20-50.

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OncoScan CNV & Oncoscan CNV Plus Assay Kit can detect mosaic aberrations found in 25% of the aberrant cells. TuScan operates in two modes:

When the percentage of aberrant cell fraction is ≥40% in a homogeneous tumor, TuScan makes calls that reflect the integer copy number in the aberrant cells.
In all other cases, TuScan makes calls down to 25% aberrant cell fractions. These are “fractional” copy number calls (such as 2.3) reflecting the average CN across the aberrant and non-aberrant cell fraction.

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The genome-wide resolution of LOH genome wide is less than or equal to 10 MB. If the aberrant cell fraction is high, the resolution increases to 3 to 5 MB.

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OncoScan CNV & Oncoscan CNV Plus Assay Kit have a 50 kb-100 kb resolution in approximately 900 cancer genes.
Outside of the cancer genes:
88% of the genome has 300 kb resolution
97% of the genome has at least 380 kb resolution

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