Focus images on the MiniSeq and NextSeq 500/550

At the beginning of Read 1 on the MiniSeq and NextSeq 500/550, the instrument will determine the best Z position (distance between the objectives and flow cell) where the clusters are well-focused. During this process, the instrument will take and save a series of Focus Images. Many of the images will be completely back, though some will show the clusters (clusters are visible in the images 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

Normal cluster density ~220 k/mm2

Standard clustering

Ultra low cluster density

All cameras disabled at cycle 1 Visible background between clusters White edges due to increased intensity

Very low cluster density

All cameras disabled at cycle 1 Visible background between clusters

Low cluster density

Low intensity or high G base calling Visible background between clusters No white edges

High cluster density

No white edges Visible fold-like artifacts

No clusters

White edges indicate increased intensity to compensate for initial lack of clusters

For any feedback or questions regarding this article (Illumina Knowledge Article #6325), contact Illumina Technical Support techsupport@illumina.com.

Last updated

© 2023 Illumina, Inc. All rights reserved. All trademarks are the property of Illumina, Inc. or their respective owners. Trademark information: illumina.com/company/legal.html. Privacy policy: illumina.com/company/legal/privacy.html