Copy Number Variation (CNV) Support—Getting Started
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The |z-score|(absolute z-score) is a statistical measurement that indicates the number of standard deviations an observation is above or below the mean of a normally distributed population. The z-score is examined when the confidence call for a sample copy number is high, e.g., above 95%. When the confidence is below 95%, it is not necessary to review the |z-score|. The |z-score| shows how meaningful the copy number call is; i.e., shows if the sample deviates significantly from the copy number mean for other samples having the same copy number call. The following guidelines below are recommended for sample copy number calls having a confidence value > 95%, if the |z-score| is: <1.75, trust the copy number call (pass); between 1.75 and 2.65, the call is borderline (pass with caution); >2.75, do not trust the call (fail).
A calibrator sample is one in which the copy number of the target gene is known. It is helpful to have for analysis, but if you do not have one the CopyCaller™ Software can still do the analysis. If a calibrator sample is not present, the software will apply an algorithm that calculates copy number assignments that maximizes the likelihood of the observed data with respect to a theoretical model.
DGV stands for Database of Genomic Variance, which is an online database of human genomic structural variation information. When applicable, every TaqMan Copy Number Assay will list the DGV variant ID that the assay is target, and links out to the database.
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.
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 on how to find a calibrator sample for CNV experiments for some screenshots on what to look for in DGV.
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.
We recommend that you to run 4 replicates per sample for reliable copy number calls.
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.
In your instrument software choose “Absolute Quantitation” or “Quantitation – Standard Curve” from the experimental properties. For analysis, we recommend to use an automatic baseline and a manual threshold of 0.2.
We offer two reference assays for human: 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.
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].
The TaqMan Copy Number Assays must be run in duplex for good quantitation results. Inclusion of the reference assay in the same reaction as the copy number assay normalizes for the amount of DNA in that particular replicate well. The CopyCaller™ Software cannot analyze data from a plate running singleplex assays.
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.
The recommend input is ~20 ng of genomic DNA. Make sure the gDNA is accurately quantitated, and to use the same amount for each sample run with the same assay.
For Research Use Only. Not for use in diagnostic procedures.