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. Configure the output folder (refer to How to connect benchtop instruments and Local Run Manager to a network location).

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:

• NextSeq 500/550 System Denature and Dilute Libraries Guide

• PhiX loading concentrations for validation runs on Illumina sequencing platforms

For any feedback or questions regarding this article (Illumina Knowledge Article #1532), contact Illumina Technical Support [e-mail].