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 - Standard Workflow | 250 pM*** | N/A** |
NovaSeq - XP Workflow | 100 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.
For any feedback or questions regarding this article (Illumina Knowledge Article #1536), contact Illumina Technical Support techsupport@illumina.com. |
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