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|
|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: