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 the sample sheet for a PhiX validation run for the MiSeq system using Illumina Experiment Manager (IEM) v1.19.1.

1. Open Illumina Experiment Manager and select Create Sample Sheet.
2. Select the MiSeq instrument and select Next.
3. Select Other.
   


4. Select FASTQ Only and then Next.
   
   • Illumina Experiment Manager User Guide
   • MiSeq System Denature and Dilute Libraries Guide
   • PhiX loading concentrations for validation runs on Illumina sequencing platforms






Run length and parameters vary depending on troubleshooting needs and reagent chemistry version. The recommended minimal read length is 26 cycles for Read 1 and Read 2 if using a MiSeq v2 50 cycle kit.

Note: some older versions of Illumina Experiment Manager do not have separate Library Prep Workflow and Index Adapters dropdown menus. For versions with only one Library Prep Kit dropdown menu, select “TruSeq LT” as the workflow.

For more information about Illumina Experiment Manager and preparing PhiX for the MiSeq sequencing platform, see the following documents: