# How to set up a PhiX validation run on the NextSeq 500/550 in manual mode

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, C, G, and T nucleotides. Illumina has currently two PhiX products available.

* **PhiX control v3 Library** is the recommended PhiX to perform validations runs. For further information, see [What is the PhiX Control v3](https://knowledge.illumina.com/library-preparation/general/library-preparation-general-reference_material-list/000001545)
* **PhiX Indexed Control**. This PhiX is not recommended to perform validation runs, as specific loading guidelines are only available for MiSeqi100. For further information, see [PhiX Indexed Control (1000 Cycle) Product Information](https://knowledge.illumina.com/instrumentation/general/instrumentation-general-reference_material-list/000009867)\
  Below are instructions for setting up a PhiX validation run for the NextSeq 500/550 system in Manual mode. Manual mode allows you to setup the run without using Local Run Manager or BaseSpace.

1. Open the NextSeq 500/550 Control Software (NCS).
2. Select **Experiment**.
3. Select **Sequence**.
4. Select **Manual**.
5. Edit the run parameters in **Run Setup**; an example paired-end run is shown below.\
   ![](https://761066130-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FGM9W2DuBTgEXv1ClCm8H%2Fuploads%2Fgit-blob-b6be147893bca00f72e25b7199ef80a61744deb5%2Fimage1.png?alt=media\&token=4c36916e-ee4c-4daf-84a7-8dca9f3e0ea7)

**Note:** For the run, set both Index 1 and Index 2 to zero. Example above is when using a 150 cycles kit. Some validation runs aim to test the overall capacity (stress test), while others analyze clustering efficiency. Adjust read length according to the purpose of the validation run and kit type.

6. Configure the output folder (refer to [How to connect benchtop instruments and Local Run Manager to a network location](https://knowledge.illumina.com/instrumentation/general/instrumentation-general-reference_material-list/000001934)).
7. Follow the prompts on the screen to load the flow cell.
8. Follow the prompts on the screen to discard spent reagents, reload the spent reagents tray, and load a new buffer cartridge.
9. Load the reagent cartridge.
10. Review the run configuration.
11. Next, perform the instrument self-check, after which the PhiX validation run begins sequencing.

For more information about preparing PhiX for the NextSeq 500/550 sequencing platform, see the following documents:

* [NextSeq 500/550 System Denature and Dilute Libraries Guide](http://support.illumina.com/downloads/nextseq-500-denaturing-diluting-libraries-15048776.html)
* [PhiX loading concentrations for validation runs on Illumina sequencing platforms](https://support.illumina.com/bulletins/2016/10/phix-loading-concentrations-for-validation-runs-on-illumina-sequencing-platforms.html)

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