Introduction

Endpoint Genotyping normally uses two differently labelled hydrolysis probes that each bind to a target which is an allele for a specific gene of interest. During amplification each probe will be incorporated into an amplicon containing their specific allele, whilst at the same time releasing fluorescence at their specific wavelength. Once complete the ratios of fluorescence is analysed to determine which allele the hydrolysis probes were binding to. These ratios are used to identify whether your samples are Wild Type, Mutant or Heterozygote.

Performing Endpoint Genotyping

To demonstrate endpoint genotyping analysis, we will use an example data file. Open the “MyGo Pro Endpoint Genotyping.ppf” file from the MyGo Pro demo data folder.

Samples Setup

Click the Samples tab to display the samples and targets for the experiment:

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Endpoint genotyping requires at least two targets be defined, each target representing one of the two variants to be identified.

Two Color Assay

Often, a two color assay will be used, with a different dye for each of the two targets. This allows probes for both targets to be present in each well. The example Experiment uses this system, and so there is only one well for each sample.

One Color Assay

However it is also possible to use a single dye to label both targets. In this case, there must be two replicates of each sample. The first replicate will contain probes for the first target, and the second replicate will have probes for the second target.

Select Analysis Type

Click the Analysis tab, Manual then add an Endpoint Genotyping analysis from the Select Analysis Type window. 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 three additional columns.

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Endpoint Genotype Calls

The Results as Table shows a genotype call for each sample that can be analysed. The column displays Allele 1 (green background) for samples showing amplification only in Target 1, Allele 2 (red background) for samples showing amplification only in Target 2, Heterozygous (yellow background) for samples showing amplifications in both targets, and N/A (grey background) for samples showing amplification in neither target.

Actual Endpoint Fluorescence (EPF)

Finally, the Results as Table display has a pair of columns showing the actual endpoint fluorescence (EPF) values for Target 1 and Target 2.

Samples as Plate

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

Settings

The pane in the top left of the window shows the settings for the Endpoint Genotyping.

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Targets

First, click the Targets tab if it is not already selected. The Targets pane allows selection of the targets to be used for the endpoint analysis. Two different targets must be selected, representing the two different variants to be genotyped. To select a target, click the drop down control, and select an entry from the displayed list of targets in the experiment.

Thresholds

The samples are classified into different genotypes by their position relative to thresholds on the scatter plot. Click the Thresholds tab:

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Minimum Angle

The first value is for Min Angle: in degrees. Any points below a line through the origin at this angle will be classified as homozygous in Allele 1. The angle is also displayed as a line on the scatter plot.

Maximum Angle

The second value is for Max Angle: in degrees. Any points above a line through the origin at this angle will be classified as homozygous in Allele 2. The angle is also displayed as a line on the scatter plot.

Minimum Radius

The third value is for Min. Radius:. Any points within this radius of the origin of the graph will be classified as NA - no amplification.

Heterozygote Classification

Any remaining points which lie outside the Min Radius: and between the Min Angle: and Max Angle: will be classified as heterozygous.

Cycles - Dye Intensity Controls

The Cycles tab contains settings for the cycles of amplification that are used to calculate the endpoint value for the targets. Click the Cycles tab:

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Endpoint Value

The endpoint value is calculated as the endpoint dye intensity, minus the background dye intensity.

First and Number of Background Cycles

Background dye intensity is calculated using the mean intensity of the dye starting from the cycle index entered in the First Background Cycle, and including the number of cycles entered in the Number of Background Cycles. So in the example experiment, there are 3 background cycles, starting from the 4th cycle and running to the 6th cycle.

Endpoint Dye Intensity

The endpoint dye intensity is calculated as the mean of the dye intensity over the last cycles of amplification. The number of cycles to average is given by the Number of Endpoint Cycles: number control.

Endpoint Graph

Click the Endpoint Graph tab, if it is not already selected. This displays a scatter plot of the levels of the two selected targets. Each point represents a sample, whose x coordinate is the endpoint level of Allele 1, and whose y coordinate is the endpoint level of Allele 2. The points are drawn differently for each genotype. Each point has a square outline, but this is filled in differently according to genotype - homozygous Allele 1 has the right hand side of the box filled in. Homozygous Allele 2 has the left side filled in, heterozygous has both sides of the box filled in, and samples with no amplification have an empty box:

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The points are hence clustered by the genotype of the samples - the cluster in the top left of the plot is for samples that are homozygous for allele 2. The cluster in the top right is heterozygous, and the cluster in the bottom right is homozygous for allele 1. The cluster near the origin, in the bottom left, consists of samples that have not amplified in either target.

Display Controls

For this section click on the Thresholds tab. The Endpoint Graph displays a scatter plot of sample’s endpoints, as described above. It also has standard controls for zooming, selecting points, and exporting data.

image alt textManual Threshold Editing

In addition, the Endpoint Graph can be used to directly edit the angle and radius thresholds for the endpoint analysis. To edit thresholds, first select the icon for threshold editing mode, to the right of the graph:

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The icon will now have a blue border, indicating that threshold editing mode is active.To edit a threshold, it must first be selected. Click the mouse on the upper line indicating the maximum angle threshold to select it:

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To move the threshold, move the mouse over the line, within the grey area, and hold down the left mouse button. Drag the mouse to place the threshold as desired, and release the button.

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The maximum angle threshold is moved on the graph, and the value is also updated in the Max Angle: number control of the Thresholds pane.The other thresholds can be edited in the same way - first click on the line to select the threshold, making it blue. Then drag the threshold with the left mouse button to move it.

Samples as Plate

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

Results as Plate

Click Results as Plate to see a display of the genotype called for each well, colored by the same code as the Results as Table display:

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