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View additional product information for CytoScan™ HT-CMA 96F Assay Kit - FAQs (906025)
25 product FAQs found
Copy Number QC Metrics:
a. SNPQC ≥ 10 - SNPQC is a measure of how well genotype alleles are resolved in the microarray data.
b. MAPD ≤ 0.28 - A global measure of the variation of all microarray probes across the genome. It represents the median of the distribution of changes in Log2 Ratio between adjacent probes. Since it measures differences between adjacent probes, it is a measure of short range noise in the microarray data.
c. Waviness SD ≤ 0.07 - A global measure of variation of microarray probes that is insensitive to short-range variation and focuses on long-range variation.
Genotyping QC Metrics:
a. DQC ≥ 0.88 - DQC is a single-sample measure of "interference" between foreground and background signal distributions. It is independent of other samples in the analysis run. Values range from 0 to 1, with higher values being better. Water is ~0.05 and 'bad' sample or assay problems center around 0.5.
b. QC Call Rate ≥ 98.5% - A metric computed over a representative set of well-behaved probe sets. Low QC call rate over well-behaved probe sets suggest problems with sample quality. Samples with low QC call rate but high DQC values are consistent with sample contamination. Monitor DQC values by plate and time to detect problem trends and problem plates.
SMN metrics:
a. MAPD (SMN) ≤ 0.35 - A global measure of the variation of all microarray probes across the genome calculated during SMN copy number analysis.
b.MAPD (SMN) ≤ 0.35 - A global measure of the variation of all microarray probes across the genome calculated during SMN copy number analysis.
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No, HTCMA and 750k have different array designs. Some regions have higher probe coverage on HTCMA than on 750k and vice versa, so some aberrations may be called differently (such as different break points).
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For unverified content, the reporting on variants is a user's decision. Users can do cross-platform testing, for example TaqMan assay/Sanger sequencing to verify the mutation.
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While the Cytoscan HT-CMA assay covers >180 structural and sequence variants, some of the mutations are extremely rare. Therefore, it is extremely challenging to get the positive clinical samples to verify the content of all the variants.
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The CytoScan HT-CMA 96F Assay Kit does not include a control. A positive control should be identified by the user and used during training and proficiency runs to generate a baseline for the control's performance. Ref103 can be purchased separately if desired (Cat. No. 951957).
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When a sample fails QC metrics, we recommend re-processing the sample starting from gDNA. Sample recovery is not possible. For example, when the WGA yield fails for a subset of samples, the gDNA quality and/or quantity should be re-evaluated before proceeding with the target prep assay. And also ensure that there are no aspirate/dispense errors in specific wells.
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The software requires that CEL files (generated from the assay run) are processed using the Best Practices Workflow option available within the software analysis configuration. This process generates QC, genotyping, copy number, and SMN results which can be opened in the Multi Sample Viewer and ChAS 4.1 Software.
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Yes. There are various options for exporting data. Please refer to the ChAS and/or RHAS User Guides:
- RHAS userguide: http://assets.thermofisher.com/TFS-Assets/GSD/Technical-Notes/rhas-1-0-user_guide.pdf
- ChAS userguide: http://assets.thermofisher.com/TFS-Assets/GSD/Technical-Notes/chas-4-1-user-guide.pdf
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This is a free download from the website: https://downloads.thermofisher.com/RHAS_1.0.zip
For processing the CEL files, a 64-bit computer with a minimum of 8 GB of RAM is required. The recommended system specifications are included in the user guide.
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For a full list of required materials, see the CytoScan HT-CMA Site Preparation Guide (manual or automated workflow). Below is a high-level account of what is required:
a. CytoScan HT-CMA Assay Kit (Cat. No. 906025) - Contains reagents, arrays, and GeneTitan MC consumables to process one 96 sample array plate.
b. GeneTitan MC Instrument (Cat. No. 00-0373) - Array plate processing occurs on the GeneTitan MC Instrument.
c. ChAS and RHAS Software: The new RHAS software is required to setup the analysis and review the data in a high volume way while ChAS is used for deeper, single sample analysis.
RHAS Software: https://downloads.thermofisher.com/RHAS_1.0.zip
ChAS 4.1 Software: https://downloads.thermofisher.com/Chromosome_Analysis_Suite_4.1.zip
d. NIMBUS Target Preparation Instrument (Cat. No. 00-0401): This is required only if the user wants to automate the sample processing (from genomic DNA to hybridization).
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We recommend using the entire plate for optimal performance. Accurate SMN1 performance requires a minimum of 90 blood samples.
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Here are the additional reagents needed:
a. 2-Propanol, anhydrous, 99.5% (isopropanol) (Sigma-Aldrich, Cat. No. 278475)
b. Reduced EDTA TE Buffer (10 mM Tris-HCl pH 8.0, 0.1 mM EDTA) (Thermo Fisher Scientific, Cat. No. AAJ75793AE) (https://www.fishersci.com/shop/products/te-buffer-1x-solution-ph-8-0-low-edta-molecular-biology-grade-affymetrix-usb-2/AAJ75793AE).
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The Cytoscan HT-CMA assay is sold as a bundle only.
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The CytoScan HT-CMA assay requires 100 ng genomic DNA in a 20 µl total volume.
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Genomic DNA extraction and purification methods that meet the general DNA requirements for this kit should yield successful results. Methods that include boiling or strong denaturants are not acceptable because the DNA would be rendered single-stranded and can no longer be accurately quantitated using a PicoGreen dye-based assay.
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The DNA requirements for the Cytoscan HT-CMA assay are outlined below:
a. DNA must be double-stranded genomic DNA.
b. DNA must be free of PCR inhibitors. Examples of DNA polymerase inhibitors include high concentrations of heme (from blood) and high concentrations of chelating agents such as EDTA. The genomic DNA extraction/purification method should render DNA that is generally salt-free because high concentrations of particular salts can also inhibit enzyme reactions
c. DNA must not be contaminated with other human genomic DNA sources or with genomic DNA from other organisms.
d. DNA must not be degraded.
e. DNA should have an A260/A280 between 1.8 - 2.0 (numeric rounding allowed) and A260/A230 greater than 1.5.
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The user guides can be accessed using the following links:
Manual Workflow User Guide: https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0018214_CytoHTCMA96F_UG.pdf
Automated Workflow User Guide for Hamilton NIMBUS: https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0018211_CytoScan_HT-CMA_NIMBUS_UG.pdf
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Yes, the same library files are used with different versions of GCC.
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For successful execution of the Cytoscan HT-CMA assay, it is important that equipment be properly maintained and calibrated per the manufacturer's specifications. Please be sure that centrifuges, pipettes, thermal cyclers, and Thermo Fisher Scientific equipment, including the GeneTitan MC instrument and external hybridization oven, if applicable, have had their recommended maintenance prior to running the assay. For lab equipment, it is typically a yearly calibration. For Thermo Fisher Scientific equipment, there is typically a scheduled annual preventative maintenance included with a Service Contract.
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This would largely depend on a laboratory's throughput requirements. The CytoScan HT-CMA 96F Assay Kit utilizes automated sample processing for 96 samples which minimizes the number of steps performed by technicians to improve quality and consistency. Typically, a lab with automation capability can process up to 1 - 8 x 96-well plates per week (i.e., 96-768 samples per week). This translates to approximately 5,000 to 40,000 samples per year.
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The CytoScan HT-CMA 96F Assay Kit has been validated to work with DNA derived from blood, cell lines, and amniotic fluid. Please note that for any other sample type, data quality will be highly dependent on the quality of genomic DNA and we would recommend discussing with your local technical sales specialist or field application specialist.
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The CytoScan HT-CMA 96F Assay Kit has been validated to work on the NIMBUS platform. We have not validated the assay on the Biomek i7 Automated Workstation.
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The CytoScan HT-CMA 96F Assay Kit has only been validated on the NIMBUS platform. If users want to automate the assay on a different instrument, it is up to them to validate that the assay is performing to their needs.
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The CytoScan HT-CMA 96F Assay Kit has a 96 array plate format.
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No, the CytoScan HT-CMA assay kit is only available in the 96 array plate format. 24 or 48 array plate formats are not offered.
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