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 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 performance.
It is important that a PhiX control run fall within optimal cluster densities for each platform, as listed in the following table.
Optimal Loading Concentration
Optimal Raw Cluster Density
iSeq 100
100 pM
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 500/550 High Output reagents
1.5 pM
170-220K clusters/mm2
NextSeq 500/550 Mid Output reagents
1.5 pM
170-220K clusters/mm2
NextSeq 1000/2000
650 pM
HiSeq 2000/2500 High Output v3
12 pM
750-850K clusters/mm2
HiSeq 2000/2500 High Output v4
18 pM
950-1050K clusters/mm2
HiSeq 2500 Rapid Run v2
12 pM
850-1000K clusters/mm2
HiSeq 3000/4000
NovaSeq - Standard Workflow
250 pM***
NovaSeq - XP Workflow
100 pM***
*Nondenatured pre-ExAmp concentration **Patterned flow cells consist of a nanowell with ordered wells, no variation in reported cluster density from run to run ***Final loading concentration
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 sequencing, see the following documents.
For any feedback or questions regarding this article (Illumina Knowledge Article #1536), contact Illumina Technical Support [email protected].
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