Troubleshooting vacuum failures and flow cell vacuum is no longer engaged message on NovaSeq 6000

Vacuum issues can happen at the start of a run or during a run, and have different underlying causes.

Vacuum issues at the start of the run

• If there are vacuum issues when loading a flow cell, please see this article: How to troubleshoot flow cell clamp or vacuum is unable to engage errors when loading flow cells on the NovaSeq 6000

• If the flow cell loads, a vacuum failure may present as a clustering failure due to the inability to flow through the flow cell.

Phenotype of runs with vacuum issues

• These manifest as low intensity and low Q30 at the inlet of the flow cell (pictured below).

• This is caused by flow cell quality issues or glass shards at the bottom of the flow cell.

• Glass shards on the flow cell stage may also impact flow cell vacuum engagement.

  

Troubleshooting steps:

1. Inspect flow cell glass and flow cell stage.

• If no glass shards are present, clean flow cell and flow cell stage with an alcohol wipe and a dry, lint-free wipe to remove any debris on these surfaces.

• For more information on how to clean the flow cell and flow cell stage, search for Knowledge Base article Troubleshooting NovaSeq 6000 flow cell loading issues video.

• Check for reagent leakage on or around the flow cell holder.

• If leaking is observed, contact Illumina Tech Support for the next steps.

1. Check the flow cell vacuum pressure (PSI) in NovaSeq Service Software (NVSS).

• Close the NovaSeq Control Software and launch NovaSeq Service Software (NVSS).

• Once initialized, select the Flow Cell option on the main screen.

• Load wash flow cells on Sides A and B.

• Pressure readings for wash flow cells are around -90 kPa and show a steady green check mark for the sensor.

• If pressure is low or the sensor is not steady, clean the flow cell and stage one more time (with alcohol and a lint-free wipe) and re-check pressure values.

  • If vacuum pressure is within the expected range, perform a power cycle on the instrument and proceed with the next run. - For more information on how to power cycle the instrument, search for Knowledge Base article How to Power Cycle the NovaSeq 6000 System? - If the issue reoccurs on the next run, contact Illumina Technical Support.

  • If pressure is still outside the expected range, contact Illumina Technical Support ([e-mail]) with a screenshot of the results.

For any feedback or questions regarding this article (Illumina Knowledge Article #1309), contact Illumina Technical Support [e-mail].