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.

• Optical alignment issue.

• Low intensity issue.

• Flow cell quality issue.

Troubleshooting steps:

1. Check the Alignment folder for autocenter images (most of the images will appear black, this is normal):

   1. C:\ProgramData\Illumina\NovaSeq\NovaSeqTemp[Run_Folder]\Alignment

      1. If the Alignment folder is not present, collect the Logs folder from here: C:\ProgramData\Illumina\NovaSeq\NovaSeqTemp[Run_Folder]. Send the zipped folder to Illumina Technical Support by attaching it to an email.

2. Check the autocenter images file extension:

   1. If the extension is \*.jpeg, open the autocenter images in a Microsoft program like Paint to check for clusters.

      1. 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 will 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 FTP site for review.

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

• For additional details on power cycling and system checks, search for 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 Check passes, 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].