How do I analyze my copy number data?

Product FAQ

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

Was this answer helpful?

Yes
No
Thank you for your response

How many replicates should I run in my TaqMan™ Copy Number Assay?

Product FAQ

Answer

We recommend that you to run 4 replicates per sample for reliable copy number calls.

Answer Id: E7546

Was this answer helpful?

Yes
No
Thank you for your response

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

Product FAQ

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

Was this answer helpful?

Yes
No
Thank you for your response

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

Product FAQ

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

Was this answer helpful?

Yes
No
Thank you for your response

I am working with a species other than human and mouse. What reference assay can I use for copy number analysis?

Product FAQ

Answer

First, you would need to select a reference sequence: usually a well studied gene, known to be present in 2 copies per diploid and found in a non-copy-number variant region of the genome. Next you can use Primer Express™ software to generate a VIC™/TAMRA™ probe-based assay from the sequence. The primers and probe sequences can be ordered online. A 20X stock reference assay should be made that contains 18 μM each primer and 5 μM probe. The reference assay should be tested on a number of samples (4 replicates each) to determine its performance before using it in duplex with CN assays. [Note: Primer Express™ Software comes free with any Applied Biosystems™ real-time PCR instrument].

Answer Id: E7550

Was this answer helpful?

Yes
No
Thank you for your response

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

Product FAQ

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

Was this answer helpful?

Yes
No
Thank you for your response

What master mix can I use with the Copy Number assays?

Product FAQ

Answer

TaqMan™ Genotyping Master Mix is the recommended master mix for use with TaqMan™ Copy Number Assays. TaqMan™ Gene Expression or TaqMan™ Universal Master Mixes can also be used.

Answer Id: E7547

Was this answer helpful?

Yes
No
Thank you for your response

What TaqMan™ Copy Number Assays are available now?

Product FAQ

Answer

We have predesigned TaqMan™ Copy number assays for human and mouse, and we also have the Geneassist Copy number assay workflow builder tool that you may use to submit sequences for custom designed assays. Copy number variation experiments require a reference assay run in duplex with the target assay. We offer RNase P and TERT reference assays for human, and we offer Tfrc and TERT reference assays for mouse. The copy number assays are FAM-MGB labeled and the copy number reference assays are VIC-TAMRA labeled.

Answer Id: E2728

Was this answer helpful?

Yes
No
Thank you for your response

Can the TaqMan™ Copy Number Assays be run using a Fast protocol?

Product FAQ

Answer

The TaqMan™ Copy Number Assays have not been validated with a Fast protocol. Preliminary studies have shown that Fast protocol analysis is not reliable for copy number quantitation analysis.

Answer Id: E7552

Was this answer helpful?

Yes
No
Thank you for your response

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?

Product FAQ

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

Was this answer helpful?

Yes
No
Thank you for your response

Where can I get reference samples for TaqMan™ Copy Number Assays?

Product FAQ

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.

Answer Id: E2733

Was this answer helpful?

Yes
No
Thank you for your response

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

Product FAQ

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

Was this answer helpful?

Yes
No
Thank you for your response

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

Product FAQ

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

Was this answer helpful?

Yes
No
Thank you for your response

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

Product FAQ

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

Was this answer helpful?

Yes
No
Thank you for your response

Which reference assay should I use for copy number analysis with human samples?

Product FAQ

Answer

We offer two reference assays for human samples: RNase P and TERT. The RNase P assay is the recommended first choice, with TERT being offered in the event that a chromosomal aberration in a sample results in suboptimal RNase P assay functionality. RNase P is located on chromosome 14, cytoband 14q11.2. The assay location is chr.14:20811565 on NCBI build 37. It has an 87 bp amplicon that maps within the single-exon RPPH1 gene. The TERT assay targets the telomerase reverse transcriptase (TERT) gene located on chromosome 5, cytoband 5p15.33. The assay location is chr.5:1253373 on NCBI build 37. It has an 88 bp amplicon that maps within exon 16 of the TERT gene.

Answer Id: E7549

Was this answer helpful?

Yes
No
Thank you for your response