How to set up the sample sheet for a PhiX validation run on the MiSeq system using Illumina Experiment Manager

03/30/20


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) v1.18.1.

  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.
  5. Enter FASTQ Only Run Settings
    1. Enter reagent barcode
    2. Select TruSeq Nano DNA as the Library Prep Workflow
    3. Select TruSeq DNA Single Indexes (A & B)
    4. Select 0 for the index read*
    5. Enter Experiment Name
    6. Enter the number of cycles for Read 1 and Read 2

      * It is critical that 0 (None) index reads is selected as the PhiX library does not contain an index. The inclusion of an index read may cause the run to terminate early.
  6. Select Add Blank Row at the bottom of the screen and enter sample information
  7. Select Finish and save the sample sheet as a .csv file

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: