Troubleshooting pre run check errors on the NovaSeq 6000

Note: NovaSeq 6000 pre-run check errors will not lock the Radio-frequency identification (RFID) for the consumables. Therefore, the consumables can be used on another instrument if runs cannot be started after troubleshooting errors.

If setting up two sides and only one side fails, the failed side can be canceled.

Alignment Failures* Alignment failures indicate the flow cell is not seated properly or an instrument optical system issue. The instrument checks auto-tilt and autocenter of the flow cell.

• Note: A flow cell loaded with the Xp workflow and being run on the wrong software version (v1.2 and lower) will pass RFID but fail during pre-run checks for alignment.

• Double check the version of the software installed is v1.3+ to support the Xp workflow.

• To troubleshoot all other alignment failures:

  • Select Reload, then select OK to confirm returning to the load screen.

  • Reload the flow cell, then select Run Setup.

  • Proceed again through the run setup to return to the Pre-Run Checks screen.

  • Redo the check.

  • If alignment still fails, collect the pre-run check logs from C:\ProgramData**\***\Illumina\NovaSeq\NovaSeqTemp\RunSetupLogs, and contact Illumina Technical Support to determine the next steps. - Note: \*The ProgramData folder may be hidden; to un-hide files, select the View tab in the file explorer window and make sure “Hidden items” is checked.

Disk Space Failures* Disk Space Failures indicate that local drives (Compute Engine (CE) and C) are full and/or if the network output folder does not exist or cannot be accessed.

• Disk Space failures can also indicate issues with network connection or CE connectivity.

• To troubleshoot:

  • Clear the local disk space through the Process Management tab in the NovaSeq Control Software.

  • Ensure both local drives have sufficient disk space: - Check disk space in the CE and the C drive: There should be 5.4 Gb/cycle (Dual Flow Cell) for the CE and 20 GB for the C drive. - Note: For the network output folder, the pre-run check only checks whether the folder exists, but not if there is enough space.

  • If registering an incorrect amount of space, check CE connectivity.

  • Close and re-open NovaSeq Control Software to see if it resolves.

  • If the issue persists, power cycle the instrument. For more information on power cycling, search for Knowledge Base article How to Power Cycle the NovaSeq 6000 System?

• Note: During Preventative Maintenance (PM) services or inspections, the instrument will save files to the C: drive. Over time this can cause the C: drive to get full and prevent runs starting until there is enough space.

• To free space from the C: drive: - Navigate to C:\Illumina\Install folder. - Search for .tif files in the install folder using the search bar in the top right. - Delete .tif files (these are large) to clear space.

Sensor Failures* This can be due to a compartment door being open, a consumable being incorrectly loaded, or a sensor having gone offline.

• To troubleshoot:

  • Select Retry and follow the onscreen prompts to resolve the error.

  • Power cycle the instrument and set the run up again.

  • It is likely a sensor issue if it fails after a power cycle. Contact Illumina Technical Support to determine the next steps.

System Connectivity failures* This is caused when the connection to Real Time Analysis v3 (RTA3), the fluidics system, or other connection on the instrument has been interrupted. The instrument contains a network hub to facilitate communication between the onboard single board computer (SBC) and the instrument components.

• To troubleshoot:

  • Select Retry and follow the onscreen prompts to resolve.

  • Power cycle the instrument and go through run setup again.

  • If it fails again, contact Illumina Technical Support to determine the next steps.

For any feedback or questions regarding this article (Illumina Knowledge Article #1295), contact Illumina Technical Support [e-mail].