Troubleshooting Possible Stale Image Data Detected on the NextSeq 500/550

The following warning occurs when issues arise during flow cell scanning in sequencing and can arise in imaging a single tile. It is usually reflected in poor sequencing metrics, such as overclustering, low passing filter, or a steep intensity/Q30 decline but does not cause the run to stop.

Warning Occurred: Possible stale image data detected Warning Occurred: Saturated image data detected

Troubleshooting steps:

1. Evaluate run performance.

2. Complete automatic post-run wash.

3. Perform system checks.

4. Share results with Illumina Technical Support.

For reference, search Knowledge Base articles:

• How to perform washes on the NextSeq 500/550

• How to perform a NextSeq 500/550 system check

For any feedback or questions regarding this article (Illumina Knowledge Article #1388), contact Illumina Technical Support [e-mail].