Troubleshooting “Unable to Register Fiducial” error on the NovaSeq 6000

Patterned flow cells use fiducials to align the instrument optical system to the flow cell before the first cycle of imaging.

• Fiducials are populated with clusters during cluster generation (Figure 1).

• When the instrument is unable to find these fiducials at the start of the run, it presents the error “Unable to Register Fiducial” and the run will stop.

  Figure 1. Reference image of a fiducial.

The most frequent causes of this error type are:

• A clustering failure related to library loading, fluidics, or temperature.

• Low intensity issue.

• Flow cell quality issue.

• Optical alignment issue.

Troubleshooting steps:

1. Open the run folder found in C:\ProgramData\Illumina\NovaSeq\NovaSeqTemp[Run_Folder]

   1. Check the Alignment folder for autocenter images. Most of the images may appear black, which is expected.

   2. If the Alignment folder is not present, collect the Logs folder found within the run folder: C:\ProgramData\Illumina\NovaSeq\NovaSeqTemp[Run_Folder]\Logs. Send the zipped folder to Illumina Technical Support.

2. Check the autocenter images file extension:

   1. If the extension is *.jpeg, open the autocenter images in a Microsoft program such Paint to check for clusters. Note: The image size may need to be reduced to fit the full fiducial on the screen and view the clusters.

   2. If the extension is *.tiff, autocenter images may need a specific program to be viewed that is not installed on the instrument. Contact Illumina Technical Support for instructions on how to upload the compressed Alignment folder to a secure site for review.

3. Power cycle the instrument and perform a System Check.

   • For additional details on power cycling and system checks, review the Knowledge Base articles How to Power Cycle the NovaSeq 6000 System and How to perform system checks on the NovaSeq 6000.

4. If the System Checks pass, proceed with sequencing on the instrument.

If another “Unable to Register Fiducial” error occurs after starting a new run (on the same side), it can indicate an issue with clustering or an instrument issue. Contact Illumina Technical Support for further assistance and provide the files below.

Files to collect:

• C:\ProgramData\Illumina\NovaSeq\NovaSeqTemp[Run_Folder]**Alignment**

• C:\ProgramData\Illumina\NovaSeq\NovaSeqTemp[Run_Folder]**Logs** (if the Alignment folder is not present).

• System Check results

For any feedback or questions regarding this article (Illumina Knowledge Article #1267), contact Illumina Technical Support [e-mail].