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

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.

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

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.
What can I do if the reference assay Ct values vary?

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.

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

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

  • Large variability in ∆Ct 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.
My results are different from what I expected. Is there something wrong with the assay?

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.

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?

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.

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

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.

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

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.

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