search
Illumina Connected SoftwareIllumina.comIllumina Support

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 v3arrow-up-right

  • 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 Informationarrow-up-right 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.

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.

  1. Configure the output folder (refer to How to connect benchtop instruments and Local Run Manager to a network locationarrow-up-right).

  2. Follow the prompts on the screen to load the flow cell.

  3. Follow the prompts on the screen to discard spent reagents, reload the spent reagents tray, and load a new buffer cartridge.

  4. Load the reagent cartridge.

  5. Review the run configuration.

  6. 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:

For any feedback or questions regarding this article (Illumina Knowledge Article #1532), contact Illumina Technical Support [email protected]envelope.

Last updated

Was this helpful?