Introduction

The MyGo Pro optical system allows for great multiplex performance. Multiplex optimisation and analysis are made very easy with the MyGo Pro software. Spectra View allows the user to balance probe concentrations, new Plate Displays allow for simplified target calling and dye selection tools improve the way you view your multiplex reactions.

Performing Multiplex Analysis

In order to demonstrate multiplex performance please open the “MyGo Five” experiment file from the MyGo Pro demo data folder.

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MyGo Five

MyGo Five is a 5 plex demo kit only compatible with the MyGo Pro. It demonstrates the multiplex capabilities of the instrument in an easy to use format. When set up correctly all reactions in the experiment contain all 5 probes at the same concentrations. Then 6 samples are defined by the primers they contain. One sample may only contain primers that amplify the FAM reaction, another sample may contain primers that will amplify all reactions. This will demonstrate the instruments ability to differentiate up to 5 reactions that are amplifying in the same tube, defined by different wavelengths. For more detail an experimental description is in the Experiment Summary under Notes.

Sample Setup

Select Samples from the left hand pane to view the samples setup.

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Above you can see the sample setup. We have 6 samples and 5 targets. Please select Samples as Plate.

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Here you can see that all 5 targets have been assigned to all 32 wells. Each sample has 4 replicates each.

Automatic Quantification

To analyse the results please select Auto. Quant.

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For more information about Auto.Quant. settings please refer to the section on Automatic Quantification.

Multiplex Tools

Dye Files

The dye files needed to analyse this experiment are available in your MyGo Pro software 2.1 and later.

Dye Selection Tool

Selecting dyes in the MyGo Pro software has been improved with the most recent releases. Please look at the Amplification window to the top right of Auto. Quant. analysis, as shown below:

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The Dye drop down menu is where you can select the virtual filters you wish to view. Above FAM is currently selected. Select the drop down menu now as shown below:

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By clicking a different dye the amplification graph will change according to your selection. By holding shift and selecting multiple dyes you can view multiple dyes at the same time. Below FAM, CAL 540 and CAL 610 have all been selected:

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If you wish to select all dyes then you can use shift or control keys to select all dyes. Alternatively you can click and drag across all dyes. The amplification graph will change accordingly.

Samples as Plate

The new Samples as Plate view has improved data analysis when multiplexing as it easily demonstrates which reactions are amplifying under which target using visual aids. Below is the Samples as Plate view for the MyGo Five reaction.

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The inner circle represents the sample for that well. The outer segment refers to the target/targets assigned to that well. All 24 wells have 5 targets assigned to them. The size of the outer segment refers to the copy number quantity it represents based on Cq values. The bigger the segment the lower the Cq and therefore the larger the estimated quantity of copy number that sample contains. As you can see above in sample 1 (red) only blue targets have amplified (FAM). In sample 2 (green) only green targets (CAL 540) have amplified and so on. For more detail on plate displays please refer to Plate Displays in the General section.

Model Amplifications

When used correctly the MyGo Five demo kit will generate data shown below:

FAM

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CAL 540

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CAL 560

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CAL 610

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CAL 635

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All 5 Probes

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