workflow guide - softgenetics · workflow guide. slide(s) topic 2-6 importing data and labeling...

Workflow Guide Slide(s) Topic 2-6 Importing Data and Labeling Samples 7-11 Processing Data Without an Allelic Ladder 12-23 Processing Data With an Allelic Ladder 24-30 Reviewing Size and Allele Calls 31-37 Automated Chimerism Analysis 38-40 Longitudinal Report 41-43 Adding Samples to a Project 44-45 Double Donor Analysis 46-52 Troubleshooting 53 More Information

To get started, import your data files. Navigate to File, and then Open Data.

Click Add and navigate to your raw data files.

2-Importing Files

Select your raw data files and then click Open, and then click OK in the Open Data Files window.

3-Importing Files

Shortcuts: Hold Shift to select multiple files at once, or click and then hold Ctrl and A to select every file.

The sample traces are now displayed in the Main Analysis Window. Double click on a sample file name to show or hide the trace.

4-Importing Files

Draw a box from left-to-right to zoom in on an area; draw a box from right-to-left to zoom out.

Click the Dye Color Icon: to display different dye colors.

Notice that the Donor and Recipient files are highlighted. In the View -> Preferences menu, under the Chimerism tab, you may enter keywords that the program will use to automatically recognize specific file types.

5 - Setting Sample Types

You can also manually enter a file’s type by simply right clicking on the file and mouse-ing over Set Sample Type.

6 – Setting Sample Types

Processing Data Without An Allelic Ladder You are now ready to process your data. However, the workflow of ChimerMarker differs depending on whether or not you choose to run an Allelic Ladder with your samples. The following section covers data processing Without a ladder. If you use a ladder in your analysis, please skip ahead to the next section, beginning on slide 12.

7 – Processing Data Without an Allelic Ladder


Process you samples by navigating to Project -> Run, or by clicking the green triangle icon. Note: set your sample types (donor, recipient, etc.) before processing your data.

From each drop-down menu, select your pre-calibrated Genotyping Panel, Size Standard, and Standard Color, or simply click the corresponding pre-saved template.

To create a new template, input a new template name and click the save button.


The second page of the Run Wizard includes parameters that govern how size and allele calls are made. The default settings are suitable for most analyses. Be sure to select Only Call Alleles in CHM Panel and Auto Create CHM Panel.

With these options selected, ChimerMarker will create a project-specific Chimertyping Panel from your Genotyping Panel. Give a name to this new panel in the field above.


If you aren’t using an allelic ladder, ensure that Auto Select Best Ladder and Auto Panel Adjustment are unselected. Click OK to process your data.


Now the data has been successfully processed. To begin reviewing your data, please skip ahead to the section titled Reviewing Size and Allele Calls, beginning on slide 24.

Note: If you notice that some high quality peaks aren’t being called by the software, please visit the Troubleshooting section, beginning on slide 46.

Processing Data With An Allelic Ladder The workflow of ChimerMarker differs depending on whether or not you choose to run an Allelic Ladder with your samples. The following section covers data processing With a ladder. If you don’t use a ladder in your analysis, please look over the preceding section, beginning on slide 7.

12 – Processing Data With an Allelic Ladder


Process you samples by navigating to Project -> Run, or by clicking the green triangle icon. Note: set your sample types (donor, recipient, and ladder) before processing your data.

From each drop-down menu, select your Genotyping Panel, Size Standard, and Standard Color, or simply click the corresponding pre-saved template.

To create a new template, input a new template name and click the save button.


The second page of the Run Wizard includes parameters that govern how size and allele calls are made. The default settings are suitable for most analyses. Be sure that Only Call Alleles in CHM Panel and Auto Create CHM Panel are not selected.


Select Auto Select Best Ladder and Auto Panel Adjustment. With these options selected, ChimerMarker will identify the best ladder sample for each sample file, and use it to create a better aligned Genotyping Panel.


The data has now been initially processed. The data may look as though it’s of poor quality, but this is because many peaks are flagged due to ploidy and heterozygous imbalance. At this stage, the alignment of the panel is most important.


If the ladder sample is in bold, it means that the program was able to align the bins of the genotyping panel to the peaks of the ladder sample, creating a new shifted panel.

Navigate to the Panel Editor (Tools -> Panel Editor) to save the newly created panel.


Observe that that are now two Project Panels: the original genotyping panel, and a second panel, whose bins have been aligned to the allelic ladder sample.


Save the newly created panel by clicking on it, and then navigating to File -> Save As New Panel. Give the new panel a name. After clicking OK, you should see the panel appear in the Panel Templates list.

You may also export the panel by right clicking on it and selecting Export.


Now, repeat the analysis using your shifted panel. Newly saved panels appear at the bottom of the panel dropdown list.


For the second run, select Auto Create CHM Panel and Only Call Alleles Present in CHM Panel.

With these options selected, ChimerMarker will create a project-specific Chimertyping Panel from your Genotyping Panel. Give a name to this new panel in the field above.


Finally, in the last window of the Run Wizard, deselect Auto Select Best Ladder and Auto Panel Adjustment.


Now a properly aligned Chimertyping Panel has been made and applied to the samples. The next section covers reviewing size and allele calls.

24 – Reviewing Size and Allele Calls

File Tree Electropherogram(s) Allele Report Table

The next group of slides demonstrates the process of reviewing size and allele calls, in preparation for Chimerism Analysis.

25 – Reviewing Size Calls

Sizing quality is indicated by the icon to the left of each file name. Green indicates successful size calling. Red and Yellow icons indicate that a size call requires review.

Click the Size Calibration Icon: to visualize size calls for each sample. Most size calling errors result from using the wrong size standard, or selecting the wrong standard color.

26 – Reviewing Allele Calls

During data processing, a Chimertyping panel is created and applied to your data. This panel includes bins for donor, recipient, and shared peaks only.

The program labels each bin according to its origin: R for recipient, D for donor, and D1R for shared peaks.

Recipient

Mixture

Donor


Allele calls are summarized in the Report Table. Again, a color scheme is used to denote quality. Green icons indicate the peak passed all analysis parameters. Yellow icons indicate the peak is in the check range for at least one parameter, and red icons indicate the a peak failed at least one analysis parameter.

The format of the report table can be modified extensively by clicking on the Report Settings Icon: The table can be exported as an excel or text file by clicking the Save icon:


The Report Table is linked with the Electropherogram. Simply double click on an allele call to be taken to the corresponding peak trace.

To obtain more information about an allele call, click on the Show Chart/Table Icon:



To edit an allele call, simply right click on the corresponding peak bin and choose which action you’d like to take.

Notice that the Allele table is updated to show what action the user has taken.

31 – Automated Chimerism Analysis

After reviewing your Size and Allele Calls proceed to the Chimerism Analysis Application. Select your preferred Chimerism Type and Quantification Method. The default settings are recommended for the remaining parameters. Under the Additional Settings tab, you can set a limit of detection threshold, and choose to display stutter-adjusted Chimerism results.


Overview of Chimersim Analysis Window

File Tree

Results Table Electropherogram(s)

To view multiple traces at once, click on the Multiple Sample View icon and select which additional traces to include. (These traces are displayed in addition to the currently selected trace.)


Note: Uninformative markers are highlighted in Red.

Marker specific and total average Chimerism for each sample are displayed in the central report table. For more information, use the Help dropdown menu.


Click on the Patient Information icon to input patient specific information. These details are included in the headers of print reports.


36 – Print Options

To print a standard report, click the printer icon and select Standard Print. Use the settings window to customize the report.

37 – Print Options

To print individual markers, use the Individual Marker Print. Again, use the settings window to format the report.

The Longitudinal Report will initially be blank. To add samples, use the green Check-Mark icon: Choose which samples you’d like to add to the report and click OK.

Another informative report is the Longitudinal Report. To access it, navigate to Tool -> Longitudinal Report, or click the Longitudinal Report icon:

38 – The Longitudinal Report


Once samples are added, the Longitudinal Report should look something like the chart shown below. The longitudinal report is excellent for displaying the trend of chimerism over time.

To print the Longitudinal Report, click the Print Icon: Use the Print Settings window to customize the report, and click OK. To save the report as a JPEG or PNG image, click the Save Icon:


41 – Adding Samples to Project

In ChimerMarker, projects can be saved in their entirety by navigating to File -> Save Project

Reopen a saved project by navigating to File -> Open Project

42 – Adding Samples to a Project

To add a sample to a project, navigate to File -> Add Samples To Project. The project panel and analysis parameters will be applied to the newly added sample. Chimerism results will now be displayed for the new sample, and the sample cab be added to the Longitudinal Report as well .

43 – Adding Samples to Project

To add a sample to the longitudinal report, click the green check-mark icon: Only samples that haven’t already been added to the report are displayed.

44 – Double Donor Analysis

The workflow of Double Donor Analysis is identical to that of Single Donor analysis. However, remember to identify all sample types (Donor1, Donor2, and Recipient) prior to data processing.

And of course, when the time comes select Double-Donor Chimerism Analysis instead of single donor analysis.

45 – Double Donor Analysis

Donor 1

Donor 2

Pre-Recipient

Mixture

Colored flags are used to denote bins from Donor1, Donor2, the Recipient, or some combination of the three.

In the Chimerism Analysis window, Donor1 and Donor2 chimerism results, and their corresponding errors, are calculated and displayed separately.

46 – Troubleshooting

After processing your data, you may find that some peaks or markers aren’t being called.

To investigate this further, navigate to the Panel Editor, which is under Tools -> Panel Editor


The Panel Editor displays the alignment of your samples to various panel templates. The Project Panel is the panel that was used to process your data. Click on a panel to see it superimposed on your sample peaks.


Genotyping Panel

Chimertyping Panel

Because the Chimertyping panel is derived from the genotyping panel, misalignments in the Chimertyping panel are typically the result of misalignments in the Genotyping Panel.


You can manually align markers and allele bins in the panel editor by holding Shift, and Clicking on the marker name or allele bin. Continue to hold Shift and the Mouse key, and drag the mouse left or right.

When you have shifted the marker or bin to your desired location, release the mouse key and the shift key.

Save your changes, and then reprocess your samples with the newly shifted panel.


Genotyping Panel

Chimertyping Panel

Properly aligned bins in the genotyping panel produce well aligned bins in the Chimertyping Panel


In ChimerMarker 3.0.2, you can use the Fixed Bin Width feature as an alternative solution to this problem. This option widens the bins of the Chimertyping panel to the width input by the user.

With this option selected, the program will widen the bins of a newly created Chimertyping panel. This will aid in calling slightly misaligned peaks that would have otherwise been missed.


Main Analysis Screen

Panel Editor

53 – More Information

All of the information described in this guide and more can be found in ChimerMarker’s linked User Manual. Find it under Help -> Help

Please send any questions to [email protected]

Or please call 814-237-9340

workflow guide - softgenetics · workflow guide. slide(s) topic 2-6 importing data and labeling...

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