Each Cq calling method analyses amplification curves to produce a value representing the cycle on which amplification is detectable. The absolute value of Cq is not normally used for analysis, instead Cq values of different reactions are used as relative values for further quantification. Different Cq calling methods may produce different values for the same curve, but the relative Cq values will always be consistent for a given Cq calling method.
Performing Cq Calling Methods
To demonstrate each Cq calling method, we will use an example data file. Open the “MyGo Pro FAM HEX.ppf” file in the MyGo Pro demo data folder. Now click the Analysis tab, where we can see that two different analyses have been created.
The first analysis uses the automatic method of Cq calling. Click on the Auto. Quant. analysis in the Analysis pane on the right hand side of the screen, if it is not already selected.
The settings for Cq calling methods are always displayed in the Settings tab in the top left. The 3.0 software has improved the way you select thresholds for positive/negative calling.
Select the settings tab as shown below:
To edit the positive / negative threshold line select the threshold icon to the right of the screen as shown below:
The software will automatically ask you to click on the graph to move the threshold. Now click on the graph to edit the threshold.
These lines can then be moved around to define your calling. The dots on the graph represent a PCR reaction from your experiment, with the x-coordinate representing a quality metric (related to how much the amplification rises above the noise), and the y-coordinate representing a performance metric (related to the efficiency). Any dots that are within the top right region of the graph are called as positive.
The threshold curve has the following parameters:
Q: the minimal quality (x-coordinate of the vertical segment)
P: the minimal performance (y-coordinate of the horizontal segment)
ΔP and ΔQ : size of the diagonal segment along the Performance and Quality axes respectively.
Two other parameters are shown in the table
M.A.R : Minimum amplification ratio. This setting has been retained for backwards compatibility, but will not show up in new files.
Excluded cycles : the number of excluded cycles at the beginning of the amplification.
Each target has its own calling settings, such that different targets can have different calling settings. It is also possible to show settings for different targets at once by selecting several targets in the dropdown.
Infer Decision Boundary
Below the table there is a button labeled “Infer decision boundary from sample assignments”. If several samples are assigned as negative, and several samples are assigned as standard, the software can use this information to guess a good decision boundary, which is what pressing this button will do.
Fit Point Quantification
For this section please open up the “MyGo Pro 2 fold dilution series.ppf.” You will see that two analyses modules have been added to the experiment. Please select the Fit Point Quant analysis module.
Click on the Settings tab in the top left hand corner, if it is not already selected:
This tab shows settings used by the Fit point Cq calling method to convert amplification curves to Cq values.
Like all Cq calling methods, there is one set of settings for each target, to allow for targets having different requirements. The Selected Target: drop down control is used to choose which target’s settings to edit - in this case there is only one target, named “Parvo”, and we are editing the settings for this.
The background dye intensity is subtracted from curves before analysis, to remove the effect of background fluorescence.
The First and Number of Background Cycles
The First Background Cycle: and Number of Background Cycles: controls are used to define a range of cycles used to estimate the background level of the reaction. The first background cycle number must be set so as to exclude any early cycles showing noise or unwanted features. The number of background cycles should then be chosen to ensure that only cycles showing no amplification are included. The default values will use cycles 3 to 6 for the background level, which should be suitable for most data.
Fit Point Thresholds
The Fit Point Cq Calling method is based on comparing each background-corrected amplification curve to a threshold dye intensity level. By observing the cycle where a curve intercepts the threshold, a Cq can be extracted. To improve accuracy, more than one point of amplification data can be analysed after the curve crosses the threshold, with a fitted curve being used to estimate the exact intercept to the threshold.
This calculation is made on the log-transformed curve, since the amplification is expected to be exponential. The threshold used for this Cq calculation is referred to as the Quantification Threshold.
A second threshold, the Positive Threshold is used to call curves as positive or negative - any background-corrected curve that reaches at least the Positive Threshold is called as positive, all others are called as negative.
Determining Your Threshold Values
There are two ways of determining the threshold values to be used for quantification and positive calling:
When the Automatic Threshold checkbox is selected (as by default), the thresholds will be calculated from the level of noise observed in the background cycles across all analysed curves. This is then multiplied by a required signal to noise ratio (SNR) to give a threshold.
The Quantification Threshold is set to the noise level multiplied by the value entered in the Quantification (xFold Global Noise): number control.
The Positive Threshold is set to the noise level multiplied by the value entered in the Pos./Neg. (xFold Global Noise): number control.
Amplification Graph Thresholds
The thresholds are also displayed in the Amplification graph, but cannot be edited directly, only by changing the SNR values:
When the Automatic Threshold checkbox is unselected, the thresholds are specified directly using the Quantification Threshold and Positive Threshold numeric controls. These directly specify the increase above background baseline, in relative fluorescence units:
Note that these directly specified thresholds are also displayed in the Amplification graph, and can be edited directly in the usual way, by selecting the threshold editing mode icon:
When this icon is selected (as indicated by a blue border), either threshold can be selected by clicking near the line. This will cause the selected line to become blue. The selected threshold can be dragged on the graph by positioning the mouse cursor over the threshold, holding down the left mouse button and moving the mouse. When the mouse is released, the threshold will be moved. The new value will also be visible in the Quantification Threshold or Pos./Neg. Threshold number control.
Exporting Cq Values
Cq values can be exported using the Export… button situated at the bottom right of the Results as Table pane for both Auto. Quant. and Fit Point. Quant analysis methods.
Cq Values as Plate
Cq Values as Plate shows Cq values as a heatmap across the plate. This is explained in more detail in Absolute Quantification.