How to set up a PhiX validation run on MiSeq (using software v2.6 and earlier)

A PhiX validation run confirms proper hardware and software performance of the instrument. The Illumina PhiX control library is derived from the small, well characterized bacteriophage PhiX genome and is a well-balanced library with relatively equal representation of A, T, G, and C nucleotides. The PhiX control library lacks an index and is not an appropriate tool for assessing Index Read performance. Run length and parameters vary depending on troubleshooting needs and reagent chemistry version. The recommended minimum read length is 26 cycles each for Read 1 and Read 2.

To perform a PhiX Validation run:

1. Set up a sample sheet in Illumina Experiment Manager (IEM).

2. Denature and dilute PhiX.

3. Load PhiX into reagent cartridge and start the run.

Set up a sample sheet in Illumina Experiment Manager (IEM)

Below are instructions for setting up the sample sheet for a PhiX validation run for the MiSeq system using Illumina Experiment Manager (IEM).

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.

Denature and Dilute PhiX Control

Use the following procedure to denature and dilute a PhiX library for use as a sequencing control. Follow the steps appropriate for the version of MiSeq reagent kit being used for the run. Further details may be found in the MiSeq Denature and Dilute Libraries Guide.

Chemistry	Final PhiX Concentration
MiSeq Reagent Kit v3	Dilute the denatured PhiX control to 20 pM, which produces an optimal cluster density using v3 reagents.
MiSeq Reagent Kit v2	Dilute the denatured PhiX control to 12.5 pM, which produces an optimal cluster density using v2 reagents.

Dilute PhiX to 4 nM

1. Combine the following volumes in a microcentrifuge tube.

• 10 nM PhiX library (2 µl)

• 10 mM Tris-Cl, pH 8.5 with 0.1% Tween 20 (3 µl)

1. If not prepared within 12 hours of the library preparation, prepare a fresh dilution of 0.2 N NaOH.

Denature PhiX Control

1. Combine the following volumes in a microcentrifuge tube.

• 4 nM PhiX library (5 µl)

• 0.2 N NaOH (5 µl)

1. Vortex briefly to mix.

2. Centrifuge at 280 × g for 1 minute.

3. Incubate at room temperature for 5 minutes.

Dilute Denatured PhiX to 20 pM

1. Add prechilled HT1 to the denatured PhiX library.

• Denatured PhiX library (10 µl)

• Prechilled HT1 (990 µl)

The result is 1 ml of a 20 pM PhiX library.

1. Invert to mix.

Note: the denatured 20 pM PhiX library can be stored up to 3 weeks at -15°C to -25°C. After 3 weeks, cluster numbers tend to decrease.

Dilute Denatured PhiX to 12.5 pM for MiSeq v2 runs

If using MiSeq Reagent Kit v3, no further dilution is required.

1. Add prechilled HT1 to the denatured PhiX library.

• 20 pM denatured PhiX library (375 µl)

• Prechilled HT1 (225 µl)

The result is 600 µl of a 12.5 pM PhiX library.

1. Invert to mix.

Load PhiX into reagent cartridge and set up run

• For MiSeq v3 reagents, load 600 µl 20 pM to reagent cartridge.

• For MiSeq v2 reagents, load 600 µl 12.5 pM to reagent cartridge.

• In the MiSeq Control Software, upload the sample sheet when requested in run set up.

For any feedback or questions regarding this article (Illumina Knowledge Article #1884), contact Illumina Technical Support [e-mail].