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. 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 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.

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

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

  3. Load the reagent cartridge.

  4. Review the run configuration.

  5. 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].

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