Failure to find fiducial errors on the NextSeq 1000/2000
Background: If the system is unable to detect clusters in the first cycle of imaging, the run may exit with the following error:
"A failure occurred during sequencing, and consumables cannot be reused. Before setting up a new run, power cycle the instrument per system guide instructions. If the error persists, contact Illumina Technical Support."
When seen at cycle 1, approximately 4 hours after run start, the most common root cause is a "Failure to find fiducial" error.
The NextSeq 1000/2000 completes an autocenter process in the blue channel at cycle 1 to find initial focus. With Patterned Flow Cell Technology, the NextSeq 1000/2000 uses clustered fiducials for Autocenter. Successful Autocenter images require signal present in blue channel at cycle 1.
Troubleshooting
Library Considerations
Use provided RSB+Tween 20 to dilute library to 2nM and then to loading concentration
Confirm library doesn't have a T overcall at cycle 1
If running Illumina mRNA stranded library with T over call at cycle 1, a custom recipe is required - NextSeq 1000/2000 control software version 1.2 and earlier, use the custom recipe found at NextSeq 1000/2000 Sequencing System Product Files - NextSeq 1000/2000 control software version 1.3 and later, custom recipe is integrated into instrument cloud run setup
Run Setup
Confirm protocol for loading library matches what is chosen in runparameters.xml
loaded double stranded DNA and false is listed
manual denature and dilute and true is listed
If using custom primers, confirm they are indicated in runparameters.xml
Reagent Preparation
Reagent thawed and prepared in accordance with recommendations in NextSeq 1000/2000 Sequencing System Guide
After reviewing library, run setup, and reagent preparation, contact Illumina Technical Support for further troubleshooting and provide the following
Collect run cycle logs located at /usr/local/Illumina/runs/[runfolder]
Collect the AutoCenter Images at /usr/local/Illumina/runs/[runfolder]/Autocenter
Contact Illumina Technical Support for a file transfer link and further troubleshooting
For any feedback or questions regarding this article (Illumina Knowledge Article #3549), contact Illumina Technical Support techsupport@illumina.com. |
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