The software now automatically determines peak areas based on local maximum compared to background levels. This prevents the user from having to add their own peak areas which becomes very useful with amplicons that contain multiple melting domains.
Performing Automatic Tm Calling
To perform Automatic Tm Calling open up the “MyGo Pro Auto Tm Calling.ppf” experiment file from the walkthrough data folder. Click the Profile tab and notice that to perform Automatic Tm Calling you will need to perform a melt. A melt can follow an amplification or can be performed on its own if pre-amplified tubes are available. In this case we have a High Resolution Melting phase which has preceded an amplification. The range of temperatures covered by the melt phase determines what data will be available for analysis - this range should cover the entire range where interesting melt features may occur, with some margin above and below.
Click on the Samples tab to view the samples and targets for this experiment:
The samples layout for an Auto Tm Calling experiment is generally simple - samples should be assigned as normal, and all targets will generally use the same dye. The melt peaks analysis will analyse a single target. The target can be present as an unknown, standard or negative, as appropriate for the experiment - all target types will be analysed. In the example experiment, all wells contain the same target as an unknown.
Select Analysis Type
Click the Analysis tab and add an Auto Tm Calling analysis from the Select Analysis Type window. If not already present learn how to do this in the Selecting Analysis Type section.
When a new Auto Tm Calling is added to the experiment, the software will automatically detect peak areas.
Samples as Plate
The Samples as Plate view shows the features described in Working with Plate Displays.
Results as Plate
Click the Results as Table tab, if it is not already selected. This shows the common features present in the Results as Table layout, as shown in General Table Layout. Additional columns include automatic peak areas columns.
Select the Peaks tab toward the top right of the screen. This shows a graph of the melt peaks. This contains the Auto peak areas, represented by the horizontal blue boxes, labelled “ Auto 1“* and ””Auto 2."* It also shows the Tm indicators, represented by small circles present on the melt Peak at the point where the Tm value was derived. The Display Peaks checkbox allows you to show or hide these indicators.
With “Display Peaks” unselected the Peaks pane shows the Auto Peak Areas only.
The default Auto Tm settings, shown under the Auto Settings pane on the top left hand side of the screen shows the default settings, as seen below.
Here the user can choose which target they wish to analyse.
The Signal-to-Noise Ratio refers to the ratio of a peak height compared to noise in the curve. In the example below the Signal-to-Noise Ratio is set at 6, therefore any peaks called will be at least 6 times higher than the noise present in the data set.