# 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.

\
\
\ <br>

|                                                                                                                                                                                                                                                                                                                                                                        |
| :--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------: |
| *For any feedback or questions regarding this article (Illumina Knowledge Article #1536), contact Illumina Technical Support* [*techsupport@illumina.com*](mailto:techsupport@illumina.com?subject=Question%2FFeedback%20Regarding%20Illumina%20Knowledge%20Article%20#000001536%20-%20Instrumentation%20\&body=Dear%20Illumina%20Technical%20Support,%0D%0A%0D%0A)*.* |


---

# Agent Instructions: Querying This Documentation

If you need additional information that is not directly available in this page, you can query the documentation dynamically by asking a question.

Perform an HTTP GET request on the current page URL with the `ask` query parameter:

```
GET https://knowledge.illumina.com/instrumentation/general/instrumentation-general-reference_material-list/000001536.md?ask=<question>
```

The question should be specific, self-contained, and written in natural language.
The response will contain a direct answer to the question and relevant excerpts and sources from the documentation.

Use this mechanism when the answer is not explicitly present in the current page, you need clarification or additional context, or you want to retrieve related documentation sections.