My results are different from what I expected when working with your Copy Number Variation Assay. Is there something wrong with the assay?

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Answer

Check that the reference assay is performing consistently across all samples. Check the concentration of your samples and make sure they are normalized. We also recommend you to use several copy number assays targeting the same variation to validate the data.

Answer Id: E7558

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CopyCaller™ Software gave the error “XXX.txt is not a compatible file and cannot be added to the analysis. Verify that the file format is compatible with the software.” What can I do now?

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Answer

Check your export results with the instrument software. The data columns need to be in the following order: Well, Sample Name, Target Name, Task Reporter, Quencher, CT. If needed, rearrange the columns and export the data again for CopyCaller™ Software.

Answer Id: E7559

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What are the TaqMan™ Copy Number Assays?

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Answer

TaqMan™ Copy Number Assays are designed for analysis of copy number variations (CNVs) and smaller regions in human and mouse genomes. We have pre-designed assays for human and mouse genomes, and we also provide custom CNV assay design service for other species as well. A TaqMan™ Gene Copy Number Assay consists of two primers and a FAM-MGB probe in a 20X formulation. Like gene expression assays, measurements are made in real time. However, gene copy number assays use genomic DNA as the template and are run as a duplex reaction with a VIC-TAMRA (non-primer limited) labeled reference assay, which normalize the gDNA input for every reaction.

Answer Id: E2619

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My data will not import into the CopyCaller™ Software. What could be wrong?

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Answer

In the instrument software, make sure that you selected “Results” from the export options. The data need to be set up as duplex reactions as well. If you ran the copy number assay and the reference assay in separate wells, the data will not be in the correct format.

Answer Id: E7560

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Can I use the CopyCaller™ Software if I am using a non-Applied Biosystems™ Real-Time PCR instrument?

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Answer

Yes, although the data will not be directly compatible. You can use the example files that install with the CopyCaller™ Software as a template to use with your data. You will have to copy/paste in the sample/target names and Ct values before importing into CopyCaller™ Software.

Answer Id: E7561

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Can I use the TaqMan™ Copy Number assays to determine high-copy insertions?

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Answer

Our copy number analysis by ddCT is best for up to 5 copies. This is because as you get to higher copy numbers, the fold change between copies gets smaller and smaller. For copy numbers above 5, it is best to use another method such as digital PCR or standard curve analysis.

Answer Id: E7545

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What can I do if the copy number confidence values are very low?

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Answer

Confidence values can be low for several reasons. Check your data for the following:

- Large variability in ΔCt values across the plate.
- Low number of replicates per sample (recommendation is for at least 4 replicates per sample).
- Sample copy number is high (> 3). You may want to use copy number bins instead for high copy numbers. Also keep in mind that CopyCaller™ Software is best for copy ranges of 1-5. If you have higher copy numbers, such as with transfections/transductions, it is best to use a standard curve.

Answer Id: E7557

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How do I find a calibrator sample for my qPCR experiment? Is it necessary for my experiment?

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Answer

We recommend that you check the DGV (database of genomic variants) database for your CNV of interest. Not all regions of variance have control samples available, but if there are controls for your CNV, the DGV will list some Coriell sample IDs. You can then order these control samples from Coriell. You can check out our blog post (http://lifetech-external.hosted.jivesoftware.com/community/real-time_pcr/blog/2013/08/16/how-to-find-a-calibrator-sample-for-a-copy-number-experiment) on how to find a calibrator sample for CNV experiments for some screenshots on what to look for in DGV.

Answer Id: E7544

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What can I do if the reference assay Ct values vary during copy number analysis?

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Answer

Check your pipettes for accuracy. Check the concentration of your samples and make sure they are normalized. Another possibility is that the chromosome on which the reference gene is located is disrupted in the samples you are working with. In this case, try a different reference assay.

Answer Id: E7556

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Can I use the TaqMan™ Copy Number assays for transgenic experiments?

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Answer

Yes, we have several assays designed to common reporters used in transgenic studies such as Cre, EGFP, and more. You can find the full list here - https://www.thermofisher.com/order/genome-database/searchResults?searchMode=keyword&CID=&productTypeSelect=cnv&alternateProductTypeSelect=&originalCount=&alternateTargetTypeSelect=&targetTypeSelect=cnv_mr&otherSpecies=&selectedInputType=&keyword=&sequen.

Answer Id: E7543

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When trying to open CopyCaller™ Software, the program freezes after launch and will not open. What can I do?

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Answer

In this case, you may see the blue CopyCaller™ Software screen pop up, but it will never advance beyond this screen to actually open the software. If this occurs, try the following:

- Press Control+Alt+Delete to bring up the Task Manager.
- Select the “Processes” tab, and look for a process called “javaw.exe”. End this process and then relaunch the CopyCaller™ Software.

Answer Id: E7555

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Can I use TaqMan™ Gene Expression Assays for a gene copy number study?

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Answer

TaqMan™ Gene Expression Assays with “_s1” as assay ID suffix are the assays whose primers and probes are designed within a single exon, and therefore, will detect genomic DNA. However, the application of these assays in gene copy number studies has not been validated. Other gene expression assays will not detect gDNA, let alone the region of variance.

Answer Id: E2736

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How do I analyze my copy number data?

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Answer

We recommend you to use our free CopyCaller™ Software. The CopyCaller™ Software was developed specifically for TaqMan™ Copy Number Assay data analysis. This free, easy-to use software utilizes a graphical interface and quickly calculates the possible copy numbers for a set of samples in a run. It also estimates a confidence value for each copy number call and has outlier removal functionality.

Answer Id: E7542

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What should I do when I get the CopyCaller™ Software error “Insufficient data points to estimate confidence and z-score values”?

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Answer

This message will appear if you don’t have at least 7 samples in a given copy number group. You can continue with the analysis, but just be aware that you will not get any statistics with your data.

Answer Id: E7554

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What type of samples should be run with TaqMan™ Copy Number Assays?

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Answer

These assays target regions of variance that are found in genomic DNA.

Answer Id: E2735

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