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

1. Collect run cycle logs located at /usr/local/Illumina/runs/[runfolder]

2. Collect the AutoCenter Images at /usr/local/Illumina/runs/[runfolder]/Autocenter

3. 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 [e-mail].