# 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. Illumina has currently two PhiX products available.

* **PhiX control v3 Library**, which lacks an index and is not an appropriate tool for assessing Index Read performance. This is the recommended PhiX to perform validations runs. For further information, see [What is the PhiX Control v3](https://knowledge.illumina.com/library-preparation/general/library-preparation-general-reference_material-list/000001545)
* **PhiX Indexed Control**. This PhiX is not recommended to perform validation runs. For further information, see [PhiX Indexed Control (1000 Cycle) Product Information](https://knowledge.illumina.com/instrumentation/general/instrumentation-general-reference_material-list/000009867)

It is important that a PhiX control run fall within optimal cluster densities for each platform, as listed in the following table. These recommendations are specific for **PhiX control v3.**

| Platform                                                               | Optimal Loading Concentration | Optimal Raw Cluster Density        |
| ---------------------------------------------------------------------- | ----------------------------- | ---------------------------------- |
| iSeq 100                                                               | 100 pM                        | N/A\*                              |
| MiniSeq                                                                | 1.4 pM                        | 170-220K clusters/mm<sup>2</sup>   |
| MiSeq v2 reagents                                                      | 12.5 pM                       | 1000-1200K clusters/mm<sup>2</sup> |
| MiSeq v3 reagents                                                      | 20 pM                         | 1200-1400K clusters/mm<sup>2</sup> |
| NextSeq 500/550 High Output reagents                                   | 1.8 pM                        | 170-220K clusters/mm<sup>2</sup>   |
| NextSeq 500/550 Mid Output reagents                                    | 1.5 pM                        | 170-220K clusters/mm<sup>2</sup>   |
| NextSeq 1000/2000 (Onboard, Standard chemistry, XLEAP chemistry P1/P2) | 650 pM\*\*                    | N/A\*                              |
| NextSeq 2000 (Onboard, XLEAP chemistry, P3/P4 and 600-cycle P1/P2)     | 450pM\*\*                     | 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\*                              |
| MiSeq i100                                                             | 120 pM\*\*                    | N/A\*                              |

\*Patterned flow cells consist of ordered nano wells, hence the reported cluster density from run to run is fixed. To assess clustering status, plotting %Occupied vs %PF is recommended.\
\*\*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.

* [iSeq 100 Sequencing System Guide](https://support.illumina.com/downloads/iseq-100-system-guide.html)
* [MiniSeq System Denature and Dilute Libraries Guide](http://support.illumina.com/downloads/miniseq-denature-dilute-libraries-guide.html)
* [Denature and Dilute Protocol Generator](https://support.illumina.com/downloads/denature-and-dilute-protocol-generator.html) (for MiSeq, NextSeq 500/550, NextSeq 1000/2000, NovaSeq 6000, MiSeq i100 and NovaSeq X Plus instruments)
* [MiSeq i100 Series Product Documentation](https://support.illumina.com/sequencing/sequencing_instruments/miseq-i100-plus/documentation.html)

**Note**: Quantification of the PhiX stock tube using Qubit or qPCR can be helpful to make sure PhiX is being diluted to the expected concentration.

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