> For the complete documentation index, see [llms.txt](https://knowledge.illumina.com/llms.txt). Markdown versions of documentation pages are available by appending `.md` to page URLs; this page is available as [Markdown](https://knowledge.illumina.com/instrumentation/novaseq-6000/instrumentation-novaseq-6000-troubleshooting-list/000001309.md). # 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](https://knowledge.illumina.com/instrumentation/novaseq-6000/instrumentation-novaseq-6000-troubleshooting-list/000001309) * 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. ![](/files/fACWwGUnxerK7jYpr6bW) **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.](https://knowledge.illumina.com/instrumentation/novaseq-6000/instrumentation-novaseq-6000-troubleshooting-list/000005612) * Check for reagent leakage on or around the flow cell holder. * If leaking is observed, contact [Illumina Technical Support](https://www.illumina.com/company/contact-us.html#/united-states/technical-support) for the next steps. 2. 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](https://www.illumina.com/company/contact-us.html#/united-states/technical-support) () 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](https://knowledge.illumina.com/instrumentation/novaseq-6000/instrumentation-novaseq-6000-reference_material-list/000002470) * If the issue reoccurs on the next run contact [Illumina Technical Support](https://www.illumina.com/company/contact-us.html#/united-states/technical-support). \ \ \
| | | :--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------: | | *For any feedback or questions regarding this article (Illumina Knowledge Article #1309), contact Illumina Technical Support* [*techsupport@illumina.com*](mailto:techsupport@illumina.com?subject=Question%2FFeedback%20Regarding%20Illumina%20Knowledge%20Article%20#000001309%20-%20Instrumentation%20\&body=Dear%20Illumina%20Technical%20Support,%0D%0A%0D%0A)*.* | --- # Agent Instructions This documentation is published with GitBook. GitBook is the documentation platform designed so that both humans and AI agents can read, navigate, and reason over technical content effectively. Learn more at gitbook.com. ## Querying This Documentation If you need additional information that is not directly available in this page, you can query the documentation dynamically by asking a question. Perform an HTTP GET request on the current page URL with the `ask` query parameter, and the optional `goal` query parameter: ``` GET https://knowledge.illumina.com/instrumentation/novaseq-6000/instrumentation-novaseq-6000-troubleshooting-list/000001309.md?ask=&goal= ``` `ask` is the immediate question: it should be specific, self-contained, and written in natural language. `goal` is optional and describes the broader end goal you are ultimately trying to accomplish on behalf of the user. GitBook uses it to tailor the answer towards what is most useful for that goal. The response will contain a direct answer to the question and relevant excerpts and sources from the documentation. Use this mechanism when the answer is not explicitly present in the current page, you need clarification or additional context, or you want to retrieve related documentation sections.