Applied Biosystems SeqStudio Genetic Analyzer Support—Troubleshooting

The SeqStudio cartridge has only been tested at the recommended storage conditions (2-8 degrees C). If storing the cartridge on the system, make sure that the Cathode Buffer Container is present and that the power to the instrument remains on.

The SeqStudio cartridge has only been tested at the recommended storage conditions (2-8 degrees C).

The red results indicator on the SeqStudio Genetic Analyzer can mean multiple things relating to the data quality (e.g., offscale signal, no signal, migration issues). More details about each of the issues and how to troubleshoot them are available in Appendix A of the SeqStudio Genetic Analyzer Instrument and Software v.1.1 User Guide.

When the hard drive on the SeqStudio Genetic Analyzer starts to get full, it will automatically delete the oldest run still on the system to make room for the new data.

If you have a Thermo Fisher Cloud account and connect the SeqStudio Genetic Analyzer to it, it is possible to monitor your runs remotely through the SeqStudio app. To set it up:

• Sign into the instrument with your Cloud instrument profile and PIN
• On the Home Screen, touch Settings > Instrument settings > Email Notifications
• On the Email Notifications screen, select or deselect which information you would like to receive email notifications on, then click Done.

NOTE: If you are logged into the local account instead of your Thermo Fisher Connect account, these options will not be active.

Here are possible causes and solutions:

• Poor size standard quality: Peak height uniformity is low or the fitting quality in sizing is poor.  Re-injecting the sample will sometimes resolve this issue.
• Sizecaller found broad peaks in the size standard: Something in the system or samples has slowed the migration of the peaks and made them broader. Re-injecting the sample will sometimes resolve this issue. This can also be caused by using an expired cartridge or Cathode Buffer Container. </li>
• Sizing quality value is in the intermediate range: The size standard data quality may be low. Re-injecting the sample will sometimes resolve this issue. If that fails, check for offscale data or peaks of low peak height in the raw data.
• Number of size standard peaks detected is less than what is defined in the size standard definition: Some of the smaller peaks in the sample may be masked by the primer peak or if the run time was shortened, the larger peaks may not be present.
• Analysis range is too small. Expand the range in the analysis settings.

The Basecalling Quality alert is triggered when the software is unable to basecall the data due to poor data quality. Re-injecting the sample will sometimes resolve the issue. If it does not, prepare a fresh sample. If the problem persists, please contact Technical Support at techsupport@thermofisher.com.

The reasons that the RFID tag on the consumables may not be recognized are:

• Missing/damaged labels
• RFID tag is in the wrong location
• RFID tag reader is not working

Check the labels on the cartridge and Cathode Buffer Container to make sure they are not damaged. Try to re-seat the consumables making sure that the cartridge has clicked into place after insertion and that the Cathode Buffer Container is firmly seated in the autosampler. If the error persists, insert consumables that have previously worked on the system. If the system recognizes these, please contact Technical Support. If the system does not recognize these, please contact Instrument Services to open a service call.

If you get a low QC flag on the SeqStudio Genetic Analyzer, it’s due to either weak signal or no signal being present in the sample. Possible reasons for this are:

• Sample volume was insufficient
• Sample concentration was too low
• Bubbles were present in the sample wells
• Sequencing reaction failed
• Old or expired reagents
• Degraded sample
• Poor sample cleanup

Re-running the samples will sometimes improve the results. Before doing so:

• Inspect the sample plate and make sure there are no bubbles on the bottom of the plate and that the volume is at least 10 µL for fragment analysis samples and sequencing samples that were not cleaned with BigDye XTerminator. For sequencing samples that were cleaned with BigDye XTerminator, minimum volume should be 65 µL after adding the SAM Solution and XTerminator Reagent.
• If the SeqStudio Genetic Analyzer cartridge or Cathode Buffer Container has expired, replace them prior to re-running the samples.
• If using BigDye XTerminator for the cleanup and if dye blobs are present, vortex the samples for an additional 10 mins at 1800-2000 rpm.

After checking the above, if the samples are still not present, try to run the Sequencing Standard control. If the signal is present, then the issue is most likely in generating the template DNA or the sequencing reaction. If the signal is not present, then try to replace the cartridge and re-run. If that fails, please contact Instrument Services as a service call will be required.

The QC flag for high signal strength on the SeqStudio Genetic Analyzer means that some of the peaks reached saturation (>30,000 rfu). Re-injection of the sample will sometimes fix this. If it does not, here are other potential fixes:

• Serial dilute the sample until it is no longer off scale.
• Adjust the injection time and voltage.

Samples showing poor resolution or loss of resolution on the SeqStudio Genetic Analyzer may have degraded over time. Samples are stable in Hi-Di Formamide for up to 24 hours on-instrument. If using the long run module, please limit the run to 48 samples to avoid degradation. If the samples are degrading faster than that, please ensure that the Hi-Di Formamide is less than a year old and has not gone through the freeze-thaw process more than 8 times. If using an injection solution other than BigDye XTerminator or Hi-Di Formamide, try switching to one of them for the injection solution. If the cartridge is past its expiry date, place a new cartridge on the instrument. Sample cleanup can also be a cause for poor data quality or loss of resolution.

Samples showing pull up/pull down in the SeqStudio Genetic Analyzer data could be due to:

• The dye calibration is not current or not matched to the samples.
• Samples are offscale (>30,000 rfu).
• Incorrect dye set was selected in the plate setup.

The automated spectral calibration algorithm requires high quality data in order to generate an optimized matrix. For fragment analysis, we recommend making a spectral using matrix standards prior to running the first sample. This only needs to be performed once before the initial run. If the spectral was just performed and passed, open the plate setup and verify that the correct dye set was selected for the run. 

The sequencing data from the SeqStudio Genetic Analyzer needs to be analyzed in Sequencing Analysis v.6.2 or later. If you currently have Sequencing Analysis v.6.0 software, you may download the latest version by going here.

The sequencing data from the SeqStudio Genetic Analyzer needs to be analyzed in SeqScape Software v.3.1 or later. If you currently have SeqScape Software v.3.0, you may download the patch by going here.

The sequencing data from the SeqStudio Genetic Analyzer needs to be analyzed in Variant Reporter v.2.1 or later. If you currently have Variant Reporter v.2.0 software, you may download the patch by going here.

The Fragment Analysis data from the SeqStudio Genetic Analyzer needs to be analyzed in GeneMapper Software v.5.1 or later. If you currently have GeneMapper Software v. 5.0, you may download the patch by going here.