Pharmacogenomics Support—Troubleshooting

Many problems with OpenArray® plate results can be diagnosed by examining the QC images taken at various points during a cycling/imaging run. In the QuantStudio® software, with the experiment in question open, click on “Export QC Images” in the Export tab. A set of images will be exported to the chosen folder location. Note: Export a set of images to a unique folder, as exporting another run to the same folder will overwrite the images.

The images exported are either fluorescent or reflected light images taken before, during, and after cycling. Real-time and SNP runs will produce somewhat different sets of images, though the ones useful for troubleshooting are easily identifiable in each set.

 
To view images, a free imaging program called ImageJ is recommended due to its ability to easily manipulate the images in ways that programs such as the built-in Windows® image viewers can’t. Many images will need to be adjusted for brightness/contrast in order to really be able to see what is happening. To do this, open the image in ImageJ, then open the brightness/contrast adjustment via ImageàAdjustàBrightness/Contrast (or press Ctrl+Shift+C). Then, simply click the Auto button (or adjust the sliders) until the features of interest in the image are visible.

Please follow the instructions below on how to clean the block.
Note: This screen is only present when the OpenArray® sample block is installed.

1. On the QuantStudio® 12K Flex Real-Time PCR System, open the instrument tray.
2. Remove the plate adapter, then place it on a clean, dry surface.
3. Retract the instrument tray.
4. Power off and unplug the instrument, then allow it to cool for 15 minutes.
5. Open the instrument access door.
6. Remove the sample block and heated cover, then place them on a clean, dry surface.
7. Remove possible contaminants (such as fluid residue or dust) by wiping the surfaces of the sample block, heated cover, and plate adapter with a 95% ethanol solution using a lint-free cloth.
8. Load the sample block and heated cover, then close the access door.
9. Plug in, then power on the instrument.
10. Open the instrument tray, then load the plate adapter.
11. Retract the instrument tray.
12. Touch Start Cleaning to begin the cleaning procedure, which takes 30 minutes to complete once the sample block and heated cover reach temperature.
13. When the procedure is complete, touch OK in the message to view the resulting image of the sample block.
14. (Optional) To save image to a USB drive:
    a. Insert a USB drive into an instrument USB port.
    b. Touch Save to USB.
    15. Touch Close to return to the Clean Instrument screen.
    16. (Optional) Touch Start Cleaning to repeat the procedure if necessary.

Go to C:\Documents and Settings\yourname\Local Settings\Application Data\Applied Biosystems\OpenArray® AccuFill™ System. Then delete Instrument.log and OpenArray® Plates History.csv. Note that this will remove all loading history, so you may want to save it elsewhere.

1. Log files from QuantStudio®12k Flex instrument
   • Insert a USB into the QS instrument
   • Follow the instructions in the “Tools” menu on the touch-screen
   • Download all 5 log files at once to the USB
     
     
2. Log files from QuantStudio®12k Software on client computer
   • Navigate to this path: C:\Applied Biosystems\AppData\QuantStudio® 12K Flex Software\UserData\data\log
   • Zip the entire contents of this directory
   • Repeat steps above with the following path:
     C:\Applied Biosystems\AppData\QuantStudio® 12K Flex Software\MetaData\logs

Extract the QC Images using the QuantStudio® software. Navigate to the folder where they were saved, and open the PRE-READ_CHANNEL_4.tiff and/or the POST-READ_CHANNEL_4.tiff files. These are the ROX™ images, so any missing through-holes are easily seen. See page 95 of the Pharmacogenomic Experiments User Guide.

Occasionally there may be probe cleavage and the NTC will cluster with the samples. First, check that not all the other assays in that subarray are affected. If all the other assays show the NTC clustering with samples then there may have been a sample tracking issue and it is not really an NTC. If all other assays in that subarray show NTC where expected, then check the amplification curve in the QuantStudio® software. Compare the Ct values of the NTC sample compared to the Ct values of samples. The NTC Ct values are high. So the probe has undergone cleavage but the signal is late and since the genotyping assay is an endpoint result, the NTC is shown to cluster with the samples. You can analyze using the “Reveal Traces” feature in QuantStudio® software, or import into Genotyper™ software to review the scatter plot at a lower cycle, before the spurious cleavage has occurred.

This means that the analysis software was unable to definitively assign a genotype call. This can be due to a number of factors including: a neighboring SNP, copy number variant, or sample quality issue (e.g., sample degradation, or the presence of impurities in the sample). If the result falls between two clusters, you should not assign a genotype call. These types of results would typically be characterized as “undetermined.” You can review the amplification curves and/or the real-time traces to check for “noisy” data, which can confirm these “bad” results.

You can download an example translation table from here. Use these examples as a template, and populate the corresponding assay and allele information for your assay of interest. This new file can then be imported into AlleleTyper™ Software.

No, it is not possible to determine the strand from the assay data. However, this information is typically not necessary.

Check the web browser you are using. AlleleTyper™ Software is accessed as an internet application that is optimized for use with the Microsoft® Windows® operating system running the Google® Chrome™ web browser v4.0 or later.

Check your table against one of our example tables to make sure the format is the same. For the calls, make sure you only have the bases listed that are possible for that assay. For example, if an assay is for an A/G SNP, if you have a T or C in the table by mistake then the file will not import.

Make sure you are exporting from Genotyper™ using Advanced mode.