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 (Figure 2), it presents the error “Unable to Register Fiducial” and the run will stop.

Figure 1. Image of fiducial with clusters.

Figure 2. Image of fiducial with little to no clusters.

The most frequent causes of these errors 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 here for autocenter images (most of the images will appear black): 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 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. 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.
3. Power cycle the instrument and perform a System Check. For more information, watch the How To Perform A System Health Check On The NovaSeq 6000
4. If the System Check tests pass, proceed with sequencing on the instrument.

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

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