Introduction

Tm Calling can be performed on data that has been acquired during a High Resolution Melting or a Melting Phase program. During amplification the amplicon becomes incorporated with an intercalating dye which increases fluorescence of the reaction. Upon melting, DNA denaturation releases this dye which decreases fluorescence of the reaction. This decrease in fluorescence is converted into melt curve data that is then used to generate melt peaks.

Performing Tm Calling

To demonstrate Tm Calling, we will use an example data file. Open the “MyGo Pro Tm Calling.ppf” file from the demo data folder. Now click the Profile tab, where we can see 2-Step Amplification run, including a High Resolution Melting Phase. An Amplification phase, is not necessary if pre-amplified product is available. 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.

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In the example data above, the melt follows an amplification and starts at an initial temperature of 60C (*Initial Stage, Temp.*) and proceeds to 95C (*Final Stage, Temp.*) at 0.05C/s (*Final Stage, Ramp*).

Samples Setup

Click on the Samples tab to view the samples and targets for this experiment. 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.

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Select Analysis Type

Click the Analysis tab and select the Manual analysis tab. From here, select Tm Calling. If not already present learn how to do this in the Selecting Analysis Type section.

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Results as Table

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, with additional fields for Peaks.

Samples as Plate

The Samples as Plate view shows the features described in Working with Plate Displays.

Melt Peak Columns

The additional columns contain peak data with a separate column for each peak region, named after that peak region. Each column displays the melt temperature, Tm, of any peak identified in the respective region and well. Where no peak is present, the cells are blank.

Exporting Melt Peak Data

The data may be exported to a Comma Separated Value (.csv) file, using the Export As… button.

Settings

Click on the Settings tab towards the top left, if it is not already selected.

image alt textMelt Peak Target Drop Down Control

This allows you to select any target in the experiment for analysis. Each melt peaks analysis is applied to a single target. If there are multiple targets of interest, then a new melt peaks analysis can be created for each one.

Use Negative Derivative

When selected, the analysis uses the negative derivative (slope) of the melt dye intensity for analysis. This is the default, and is suitable for most applications. When the checkbox is not selected, the analysis uses the positive derivative, which is suitable for protein melts, etc.

Noise Reduction Range

This specifies the expected, approximate width of melt peaks in the data. The default value of 1C will be suitable for most applications. Increasing the peak width allows for better identification of broad peaks in lower-quality data, but will limit the resolution of the analysis. Decreasing the peak width allows for distinguishing narrower peaks in good quality data, but will increase the apparent noise level.

Peak Areas

This shows a table, containing the regions where melt peaks will be extracted from the data. There are three peak regions already defined - “Peak 1” to “Peak 3”.

Click on the Peak Areas tab:

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Peak Area Temperature Ranges

For each peak region, two temperatures are defined - Temp.1 and Temp. 2. these define the range of temperatures where a peak is expected to occur. Temp. 1 is commonly used for the minimum temperature in celsius, and Temp. 2 for the maximum temperature, but the two temperatures need not be in order - Temp. 2 may be lower than Temp. 1, in which case Temp. 2 will be used as the minimum.

Peak Area Dye Threshold

For each peak region, a dye intensity threshold is defined - only peaks which have a greater intensity will be extracted.

Editing Peak Areas

The peak region names, temperatures and threshold can be edited directly in the table if desired, and the standard “+”, “-”, and up/down arrow buttons can be used to add, delete and move the peak regions. As many peak regions as required can be added, subject to PC performance.

Melt Peaks Graph

This chart displays the derivative of the dye intensity with temperature, in the profile’s melt phase. As can be seen in the example Experiment, this produces characteristic peaks showing the rate of melting of products.

Click the Melt Peaks tab if it is not already selected:

image alt textMelt Peak Regions

Overlaid on the graph are region displays, showing the areas of the graph where peaks will be extracted. Each peak region is displayed as an “H”, where the vertical lines denote the minimum and maximum temperature, and the horizontal bar denotes the intensity threshold. The name of the region is displayed alongside the minimum temperature line. A shaded region in the upper half of the “H” shows the area of the graph where the melt must exhibit a peak, in order for the peak to be extracted by the analysis.

Display Controls

The graph has standard controls for zooming, selecting curves, and exporting data.

Selecting and Moving Peak Regions

The graph has a control for selecting and dragging the peak regions. This is represented by an icon resembling the peak region displays on the graph itself. Click this icon to select it, if it is not already selected.

To select a peak region, click on any of the lines of the region - the horizontal line, or either of the vertical lines. This will change the color of the selected region from grey to blue. Note that the region will also be selected in the Thresholds pane - either the graph or the table can be used to make your selection.

Editing a Peak Region

To edit a peak, move the mouse over the line you wish to move, and hold down the left mouse button. Drag the line to the desired position, and release the left mouse button. Note that only the selected region can be dragged - to edit another region, click it first to select it (making it blue), then drag to edit.

Melt Curve

Click on the Melt Curve tab:

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This graph shows the raw dye intensity (in Relative Fluorescence Units, RFU) against temperature in celsius. The graph has standard controls for zooming, selecting curves, and exporting data.

Notes

Click on the Notes tab:

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This tab displays any information, warnings and/or errors generated by the analysis. In this case there are no notes.

Sample as Plate

The Samples as Plate view shows the features described in Working with Plate Displays.

Tm as Plate

This displays a heatmap of the melt temperatures for a selected peak region. The display is laid out in the same physical pattern as wells in an instrument, with each well colored according to the temperature of any extracted peak, or grey where there is no target or peak present.

Click the Tm as Plate tab:

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Peak Area for Plate Display

To the right of the plate display are the controls for the display:

Selected Peak Area

This drop down control is used to select any of the peak regions defined in the analysis. Only a single peak region can be displayed at a time. The wells are colored according to a rainbow scale from blue for the lowest Tm to red for the highest.

User-specified range

When selected, the user can determine the temperature range at which the Tm will be displayed in. The range will become more accurate the closer you come to the range specified in Selected Peak Area. Only one peak area can be analysed at one given time.

Range from peak range

The coloured Tm display will now correspond to the peak chosen in Selected Peak Area only. The temperature range can not be modified in this mode.

Range from selection

This allows the user to view a Tm display based on the wells that have been chosen in the plate. The software will calculate the maximum and minimum temperatures of the selection and derive the Tm range display from that.