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.

1. 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.

2. Phenotype of runs with vacuum issues

• When reviewing the run data in Sequencing Analysis Viewer (SAV), these manifest as low intensity and low Q30 at the inlet of the flow cell (see Figure 1).

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

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

  

Figure 1: Flow Cell Chart displaying the detected intensity in SAV.

Troubleshooting steps:

1. Inspect flow cell glass and flow cell stage.

• If glass shards or debris are found, blot the flow cell glass or flow cell stage with an alcohol wipe to pick up the debris.

• Once no debris is identified, clean the flow cell and flow cell stage with a lint-free alcohol wipe and dry with a low-lint lab tissue.

  • For more information on how to clean the flow cell and flow cell stage, please review the 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 Technical Support for the next steps.

1. Check the flow cell vacuum pressure in NovaSeq Service Software.

• Close the NovaSeq Control Software and launch the NovaSeq Service Software.

• Once initialized, select Load Flow Cell.

• Load wash flow cells on Sides A and B.

• Expected Vacuum Pressure Values for wash flow cells are around -90 kPa and show a steady green check mark for the Vacuum Pressure State.

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

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

• If vacuum pressure is within the expected range, perform a power cycle and proceed with the next run.

  • For more information on how to power cycle the instrument, review the Knowledge Base article How to Power Cycle the NovaSeq 6000

  • If the issue reoccurs on the next run contact Illumina Technical Support.

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