# 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](https://761066130-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FGM9W2DuBTgEXv1ClCm8H%2Fuploads%2Fgit-blob-ab7dd7eb4dc1080b1c8b1dc933b5c5c3198129ce%2Fimage1.png?alt=media)                                                                                                                                                                                                                                                                                 |
| 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](https://761066130-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FGM9W2DuBTgEXv1ClCm8H%2Fuploads%2Fgit-blob-3d21527fa848e6309690d8f3dfb2882442dfa491%2Fimage2.png?alt=media)                                                                                                                                                                                                                                                                            |
| Very low cluster density                     | <p>All cameras disabled<br>at cycle 1<br><br>Visible background between clusters</p>                                               | ![Very low cluster density.jfif](https://761066130-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FGM9W2DuBTgEXv1ClCm8H%2Fuploads%2Fgit-blob-3636296ee53d36a23193f484737f75c62b8a1df6%2Fimage3.png?alt=media)                                                                                                                                                                                                                                                                     |
| 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](https://761066130-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FGM9W2DuBTgEXv1ClCm8H%2Fuploads%2Fgit-blob-1b43720d6208e49edba4b5b0c6255b68b21cfcda%2Fimage4.png?alt=media)                                                                                                                                                                                                                                                                          |
| High cluster density                         | <p>No white edges<br><br>Visible fold-like artifacts</p>                                                                           | <p><img src="https://761066130-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FGM9W2DuBTgEXv1ClCm8H%2Fuploads%2Fgit-blob-a41def6deb18eb14ba69338aed0b4df0bdaddbb5%2Fimage5.png?alt=media" alt="High cluster density 1.jfif"><br><img src="https://761066130-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FGM9W2DuBTgEXv1ClCm8H%2Fuploads%2Fgit-blob-2f05cc474362cccaad66f1af66fb208cc2a408bd%2Fimage6.png?alt=media" alt="High cluster density 2.jfif"></p> |
| No clusters                                  | White edges indicate increased intensity to compensate for initial lack of clusters                                                | ![No clusters.jfif](https://761066130-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FGM9W2DuBTgEXv1ClCm8H%2Fuploads%2Fgit-blob-3019f588dfa587768d6d0065d289c240adc1b49a%2Fimage7.png?alt=media)                                                                                                                                                                                                                                                                                  |

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