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
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 [e-mail].