# Focus images on the MiniSeq and NextSeq 500/550

At the beginning of Read 1 on the MiniSeq and NextSeq 500/550, the instrument determines the best Z position (distance between the objectives and flow cell) where the clusters are well-focused. During this process, the instrument takes and saves a series of Focus Images.

Many of the images are black with a laser dot and contain in the file name nomenclature, "Laser\_AF\_zpos... method\_AutoFocus". These images are laser only and thus clusters are not excited and not seen.

However, some focus images show clusters and have in the file name nomenclature, "Red\_IM\_zpos... method\_AutoFocus\_BestFocus". These images show visible clusters when the Z position is correct, allowing for proper focus of the clusters on the flow cell.

Technical Support can evaluate Focus Images to diagnose clustering issues (failed to cluster/ underclustering and overclustering) or an unstable focus model.

* Location of Focus Images:
  * MiniSeq: D:\Illumina\MiniSeq Sequencing Temp\[Run Folder]\Images\Focus
  * NextSeq 500/550: D:\Illumina\NextSeq Control Software Temp\[Run\_Folder]\Images\Focus

Example Focus Images:

| **Clustering**                               | **Phenotype**                                                                                                                      | **Example Image**                                                                                                                                             |
| -------------------------------------------- | ---------------------------------------------------------------------------------------------------------------------------------- | ------------------------------------------------------------------------------------------------------------------------------------------------------------- |
| <p>Normal cluster density<br>\~220 k/mm2</p> | Standard clustering                                                                                                                | ![rtaImage (1).jfif](/files/GPX61ZK7Xl0P7SfJmtQV)                                                                                                             |
| Ultra low cluster density                    | <p>All cameras disabled<br>at cycle 1<br><br>Visible background between clusters<br><br>White edges due to increased intensity</p> | ![Ultra Low Density.jfif](/files/v5fSmuYGERmUbXMXs6sQ)                                                                                                        |
| Very low cluster density                     | <p>All cameras disabled<br>at cycle 1<br><br>Visible background between clusters</p>                                               | ![Very low cluster density.jfif](/files/vbFrzW40MyLvfIbsoWJR)                                                                                                 |
| Low cluster density                          | <p>Low intensity or high G base calling<br><br>Visible background between clusters<br><br>No white edges</p>                       | ![Low cluster density.jfif](/files/J6FRvrbrYfbeUEwtSVny)                                                                                                      |
| High cluster density                         | <p>No white edges<br><br>Visible fold-like artifacts</p>                                                                           | <p><img src="/files/BUtenyg3D3Sz27BS2QBl" alt="High cluster density 1.jfif"><br><img src="/files/ToQJ7TfmH9OE9gll8uKo" alt="High cluster density 2.jfif"></p> |
| No clusters                                  | White edges indicate increased intensity to compensate for initial lack of clusters                                                | ![No clusters.jfif](/files/mo1QzmpRw5Xzyhl2HGCj)                                                                                                              |

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| *For any feedback or questions regarding this article (Illumina Knowledge Article #6325), contact Illumina Technical Support* [*techsupport@illumina.com*](mailto:techsupport@illumina.com?subject=Question%2FFeedback%20Regarding%20Illumina%20Knowledge%20Article%20#000006325%20-%20Instrumentation%20\&body=Dear%20Illumina%20Technical%20Support,%0D%0A%0D%0A)*.* |


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