> 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-reference_material-list/000002881.md).

# How to set up a PhiX validation run on the NovaSeq 6000 sequencing platform

A PhiX validation run confirms proper hardware and software performance of the instrument. The Illumina PhiX control library is a well-balanced genome with relatively equal representation of A, T, G, and C nucleotides. PhiX lacks an index and is not an appropriate tool for assessing Index Read performance. Below are instructions for setting up a PhiX validation run on NovaSeq 6000 sequencing platforms.

The optimal loading concentration for a PhiX validation run on the NovaSeq system is 250 pM. Refer to the [NovaSeq 6000 Denature and Dilute Guide](https://support-docs.illumina.com/IN/dnd-wizard/Content/dnd/landing.htm?ins=novaseq6000) for the denaturation and dilution procedure. Load denatured PhiX into the library tube just prior to run setup. The uncapped library tube is loaded into position 8 of the cluster cartridge.

* **Note:**
  1. Any NovaSeq 6000 flowcell (SP, S1, S2, or S4) can be used for a PhiX validation run; to test all four lanes, a NovaSeq S4 300 cycle kit is recommended.
  2. For S4 runs, 4 tubes of PhiX will be required per flowcell.

1. Open the NovaSeq 6000 Control Software (NVCS)
2. Select **Sequence** on the Home screen and select one or both sides.
3. Follow the prompts on the screen to load the flow cell.
   1. Note: Do not place anything on top of the flow cell door.
4. Follow the prompts on the screen to load the cluster, SBS, and buffer cartridges, and to empty the large and small waste bottles.
5. (Optional) If uploading the run to BaseSpace Sequence Hub, select **Run Monitoring Only**. If **Run Monitoring and Storage** is selected, a sample sheet will be required.
6. On the **Run Setup** screen:
   1. Enter run parameters - typically 2 x 151.
   2. Set number of cycles for Index 1 and Index 2 to zero.
7. Specify the desired network output location.
8. Review the run parameters, then proceed to the pre-run checks.
9. After the checks have successfully completed, your sequencing run will begin.

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| *For any feedback or questions regarding this article (Illumina Knowledge Article #2881), contact Illumina Technical Support* [*techsupport@illumina.com*](mailto:techsupport@illumina.com?subject=Question%2FFeedback%20Regarding%20Illumina%20Knowledge%20Article%20#000002881%20-%20Instrumentation%20\&body=Dear%20Illumina%20Technical%20Support,%0D%0A%0D%0A)*.* |


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