# How to set up a PhiX validation run on MiSeq i100 Series

**Background**

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 or DRAGEN Secondary Analysis performance.

Below are instructions for setting up a PhiX validation run on MiSeq i100 series sequencing platforms. The optimal loading concentration for a PhiX validation run on the MiSeq i100 series is 120 pM.

**Plan a Sequencing Run**

*Cloud run planning:*

To plan run in the Cloud using BaseSpace Sequence Hub, use the Run Planning tool in BaseSpace Sequence Hub to set up a sequencing run. For more information on BaseSpace Sequence Hub, refer to the [BaseSpace Sequence Hub support site page](https://support.illumina.com/sequencing/sequencing_software/basespace.html).

*Local run planning:*

1. Select the menu icon in the upper-left corner.
2. Select **Runs**.
3. On the Planned tab, select **Create run**.
4. Enter a run name to identify the run.
5. Enter the number of cycles performed in each read:
   1. For the 100 cycle kit, use 51 cycles for Read 1 and Read 2
   2. For the 300 cycle kit, use 151 cycles for Read 1 and Read 2
   3. For the 600 cycle kit, use 301 cycles for Read 1 and Read 2.
   4. For the 1000 cycle kit, use 501 cycles for Read 1 and Read 2 (the indexed PhiX Control is recommended for this setup due to its bigger insert size).
   5. Set number of cycles for Index 1 and Index 2 to 0 (zero) for all kits. Do not leave these fields blank.
6. Select **Next**.
7. Select the analysis application, typically **DRAGEN BCL Convert**.
8. Select the library prep and index adapter kits, typically **Not Specified**.
9. Select **Next**.
10. Input the Sample ID, typically **PhiX**.
11. Select the FASTQ file compression format, typically **gzip**.
12. Select **Next**.
13. Select **Save as Planned**.

**Start a Sequencing Run**

1. Select **Start**.
2. Sign in with local or cloud account.
3. Select the PhiX run configured in the steps above from the **Planned Runs** list.
4. Select **Review** and then review your run information.
5. Select **Load consumables**.
6. Place the new dry cartridge in the dry cartridge tray. Gently push the dry cartridge until it touches the back of the tray, so it is firmly secured.
7. Select **Next**.
8. Remove the plastic cap and load the wet cartridge.
9. Select **Close**.
10. Select **Verify Run**.
11. Confirm the run and consumables are correct, and then select **Start run**.

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| *For any feedback or questions regarding this article (Illumina Knowledge Article #9320), contact Illumina Technical Support* [*techsupport@illumina.com*](mailto:techsupport@illumina.com?subject=Question%2FFeedback%20Regarding%20Illumina%20Knowledge%20Article%20#000009320%20-%20Instrumentation%20\&body=Dear%20Illumina%20Technical%20Support,%0D%0A%0D%0A)*.* |


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