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.
Platform
Optimal Loading Concentration
Optimal Raw Cluster Density
iSeq 100
100 pM
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 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
N/A**
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
2-3nM*
N/A**
NovaSeq 6000 - Standard Workflow
250 pM***
N/A**
NovaSeq 6000 - XP Workflow
100 pM***
N/A**
NovaSeq X - 1.5B/10B
140 pM***
N/A**
NovaSeq X - 25B
140 pM***
N/A**
*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.
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