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

Normal cluster density ~220 k/mm2

Standard clustering

rtaImage (1).jfif

Ultra low cluster density

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

Ultra Low Density.jfif

Very low cluster density

All cameras disabled at cycle 1 Visible background between clusters

Very low cluster density.jfif

Low cluster density

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

Low cluster density.jfif

High cluster density

No white edges Visible fold-like artifacts

High cluster density 1.jfif High cluster density 2.jfif

No clusters

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

No clusters.jfif

For any feedback or questions regarding this article (Illumina Knowledge Article #6325), contact Illumina Technical Support [email protected].

Last updated

Was this helpful?