# Illumina测序平台测试Run中PhiX的上机浓度

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PhiX测试Run是Illumina用来确认测序仪硬件和软件是否可以正常工作的实验方法。这里所用的PhiX参照文库基因组碱基的平衡性很好，A，T，C，G四种碱基的比例相对均一。Illumina 目前提供两种类型的PhiX产品可供用户选择。

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**PhiX Control v3** , 该文库没有Index 不适用于Index 测序的性能评估。这种推荐用于验证运行(validations runs)。更多信息可以点击查看[PhiX Control v3文库是什么，它在Illumina下一代测序中起到什么作用？](https://knowledge.illumina.com/library-preparation/general/library-preparation-general-reference_material-list/000007130)

**PhiX** **Indexed Control**. 这种 PhiX 不推荐用于验证运行(validations runs)。 更多信息可以点击查看[PhiX Indexed Control (1000 Cycle) Product Information](https://knowledge.illumina.com/instrumentation/general/instrumentation-general-reference_material-list/000009867)

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测试时, 确保PhiX参照文库的簇密度落在最佳范围内对测试结果的准确性非常重要。请参考下面表格了解不同测序平台PhiX的上机浓度和簇密度建议（这些推荐的参数是基于**PhiX control v3** ）：\
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| 测序平台                                                                   | 最佳上机浓度     | 最佳原始簇密度                 |
| ---------------------------------------------------------------------- | ---------- | ----------------------- |
| 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                                   | 1.8 pM     | 170-220K clusters/mm2   |
| NextSeq 500/550 Mid Output reagents                                    | 1.5 pM     | 170-220K clusters/mm2   |
| 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)     | 450 pM\*\* | 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\*                   |

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\*Patterned flow cells上面都有固定量的纳米孔，所以不同的run显示的簇密度都是相同的。如需评估簇密度情况，推荐通过[绘制 %Occupied 与 %PF 关系图](https://knowledge.illumina.com/instrumentation/general/instrumentation-general-reference_material-list/000007138)查看。

\*\*为最终的上机浓度

对于PhiX测试Run，测序读长和参数可以根据实验目的进行调整，但是请注意Read1和Read2必须至少是26个Cycles，这样仪器上的数据分析软件才能生成质量分数（Quality Scores）。

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有关为Illumina测序平台准备PhiX测试Run的更多信息，请参见以下文档:

* [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)

**备注**: 推荐对Phix 原管储存液进行Qubit 或qPCR定量后再稀释到需要的浓度使用。

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| *For any feedback or questions regarding this article (Illumina Knowledge Article #7139), contact Illumina Technical Support* [*techsupport@illumina.com*](mailto:techsupport@illumina.com?subject=Question%2FFeedback%20Regarding%20Illumina%20Knowledge%20Article%20#000007139%20-%20Instrumentation%20\&body=Dear%20Illumina%20Technical%20Support,%0D%0A%0D%0A)*.* |


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