How to Spike in PhiX for the MiSeq i100 Series

Background

The PhiX v3 Control library represents an important positive control for sequencing run experiments and is useful when troubleshooting sequencing runs. Additionally, PhiX can be used to provide nucleotide diversity and balance to an otherwise imbalanced library pool, improving run quality and Yield for low diversity library. See the following instructions for spiking in PhiX when preparing libraries for sequencing on the MiSeq i100 Series.

1. Thaw the 10 nM PhiX stock solution on ice.

2. Dilute PhiX to 6x the final library loading concentration with RSB.

   1. Example: For a final loading concentration of 100 pM, dilute PhiX to 600 pM (0.6 nM).

3. Add 1 µL of the 6x PhiX solution to 30 µL of 10x loading concentration library mixture.

4. These volumes produce approximately a 2% PhiX spike-in. The percentage varies depending on the library quality and quantity.

To spike-in a higher percentage concentration of PhiX, simply divide the desired spike-in percentage by 2 to get the volume of 6X PhiX in µL and then add the remaining volume of 10x library to a final volume of 30 µL. For example:

1. For a 5% target spike-in, divide 5% by 2 for 2.5 µL.

2. Add 2.5 µL of 6x PhiX to 27.5 µL of 10X library pool for a final volume of 30 µL.

See the Table below for common PhiX spike in percentages and the expected volume of 6X PhiX to add.

Spike-in Percentage	Volume of 6x PhiX (uL)
2%	1
5%	2.5
10%	5
15%	7.5
20%	10

For any feedback or questions regarding this article (Illumina Knowledge Article #9404), contact Illumina Technical Support [e-mail].