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 either 6X or 10X the library loading concentration depending on PhiX spike-in value.

   1. For intended PhiX spike-in <10%, dilute PhiX to 6x library loading concentration with RSB.

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

   2. For intended PhiX spike-in ≥10%, dilute PhiX to 10x library loading concentration with RSB.

      1. Example: For a final loading concentration of 100 pM, dilute PhiX to 1000pM (1nM).

3. Combine appropriate volume of the PhiX solution and 10x loading concentration library mixture to create 30 µl mixture.

   1. Example 2% PhiX spike in:

      1. Add 1 µl of 6x PhiX solution to 29 µl 10x loading concentration library pool.

   2. Example 5% PhiX spike in:

      1. Add 2.5 µL of 6x PhiX solution to 27.5 µL of 10X loading concentration library pool.

   3. Example 10% PhiX spike in:

      1. Add 3 µl of 10x PhiX solution to 27 µl 10x concentration libraries to obtain 30 µl 10x library mixture.

4. The final PhiX %Aligned in the Run Metrics might not match the intended spike-in and varies depending on the library quality and quantity.

Quick Calculation Notes:

• For PhiX spike in <10%, divide desired spike-in percentage by 2, then add that volume of 6x PhiX solution.

• For PhiX spike-in ≥10%, multiply 30 µl by desired percentage and divide by 100, then add that volume of 10x PhiX solution.

Table 1: Spike-in volumes for common PhiX spike in percentages.

Spike-in Percentage	Volume of 6x PhiX (µl)
2%	1
5%	2.5

Spike-in Percentage	Volume of 10x PhiX (µl)
10%	3
15%	4.5
20%	6
25%	7.5

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