PhiX loading concentrations for validation runs on Illumina sequencing platforms


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. PhiX lacks an index and is not an appropriate tool for assessing Index Read performance.

It is important that a PhiX control run falls within optimal cluster densities for each platform, as listed in the following table.

Platform Optimal Loading Concentration Optimal Raw Cluster Density
HiSeq 2000/2500 High Output v3 12 pM 750-850K clusters/mm2
HiSeq 2500 High Output v4 18 pM 950-1050K clusters/mm2
HiSeq 2500 Rapid Run v2 12 pM 850-1000K clusters/mm2
HiSeq 3000/4000 2-3nM* N/A**
MiniSeq 1.4 pM 170-220K clusters/mm2
MiSeq v2 reagents 12.5 pM 1000-1200K clusters/mm2
MiSeq v3 reagents 20 pM 1200-1400K clusters/mm2
NextSeq High Output reagents 1.8 pM 170-220K clusters/mm2
NextSeq Mid Output reagents 1.5 pM 170-220K clusters/mm2
NovaSeq - NovaSeq Control Software v1.1 or lower 300 pM N/A**
NovaSeq - NovaSeq Control Software v1.2 or later 175 pM N/A**
iSeq 100 60 pM N/A**

*Nondenatured pre-ExAmp concentration
**Patterned flow cells consist of a nanowell with ordered wells


Run length and parameters vary depending on troubleshooting needs, however, Read 1 and 2 must have at least 26 cycles to generate quality scores.

For further information about preparing PhiX for your sequencing platforms, see the following documents: