Troubleshooting Cycle 1 Error, Camera Disabled, Failed to Detect Clusters on the NextSeq 500/550

This notification indicates that the NextSeq 500/550 is unable to detect clusters at cycle 1 for the reported cameras, lanes, and/or surfaces. A large number of sections disabled may be reported.

Potential causes for cycle 1 errors include instrument, sequencing consumables, or library (design/custom primers/quantification/denaturation) factors.

• If the run stops at Cycle 1 and Lanes 1/3 show low cluster density (no focus images for Lanes 2/4), this is likely a clustering failure; follow the troubleshooting steps below (to troubleshoot the instrument, flow cell, and library).

• If the run stops at Cycle 1 and Lanes 1/3 show good cluster density but Lanes 2/4 do not, this shows that the library clustered successfully; troubleshoot this as a flow cell or instrument issue (there is no need to troubleshoot the library in the troubleshooting steps below).

Example of error messages:

The following message may also be present:

Note 1: It is not recommended to save the flow cell for rehybridization following a cycle 1 cameras disabled error. See NextSeq 500 System Guide or NextSeq 550 System Guide Appendix A Troubleshooting: Rehybridization Workflow for more information.

Note 2: The instrument may give an option to purge reagents after the run ends. This option will proceed with purging consumables and then performing the automatic post-run wash. The purging process can take a few hours (especially for a cycle 1 error when most of the reagents are still in the reagent cartridge), to initiate troubleshooting immediately, bypass the reagent purge.

Troubleshooting steps:

1. Acknowledge the error message. The flow cell cannot be saved. Illumina does not require the purge. Perform the post-run wash.

2. Power cycle the instrument.\*

3. Perform System Check\* with a used flow cell. Illumina recommends using a high output flow cell (a high output flow cell is recommended since mid-output flow cells can have false flow rate failures).

4. Review the Focus Images:

   1. Focus images are saved even if run stopped at cycle 1 and can be found at D:\Illumina\ NextSeq Control Software Temp[Run_Folder]\Images\Focus\L00X\C1.1

   2. Images may be present for all 4 lanes or lanes 1/3 only.

   3. It is normal for many images to be black, these images are taken at different heights to find focus.

   4. See Knowledge Base Article Focus images on MiniSeq****and NextSeq 500/550 for more information.

5. If a clustering issue is suspected, troubleshoot the library:

   1. Review Library type/design.

   2. Library quantification.

   3. Denaturation steps.

   4. Use or requirement of custom sequencing primers.

\*For further information, search for Knowledge Base articles How to Perform a NextSeq 550 Power Cycle and How to Perform a NextSeq 550 System Check.

For any feedback or questions regarding this article (Illumina Knowledge Article #1681), contact Illumina Technical Support [e-mail].