08/23/2022
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 your sequencing platforms, see the following documents: