Troubleshooting Custom Sequencing Primers selected by mistake in manual mode on the NextSeq 500/550

Users who upgraded NextSeq Control Software (NCS) to v4.0 and v4.0.1 may have seen sequencing runs with steep Intensity and Q30 declines at the start of one or more reads. This can be caused by primer hybridization failures that may occur due to a mistake during run setup in manual mode.

The custom primer checkbox area was larger than the visual checkbox area, which made it prone for users enabling the option by mistake. This has been addressed in NCS v4.0.2. Illumina recommends updating to NCS v4.0.2 if possible.

The following screenshots can be useful in understanding the issue, as well as where to look during run setup (manual mode) review before proceeding with the pre-run checks. If one or more custom primer options were inadvertently selected; select the Edit option and correct the selections prior to proceeding with the pre-run checks.

Custom Primer checkboxes on screen:

Run setup review - no custom primer selected:

Run setup review - custom Read 2 primer selected:

For any feedback or questions regarding this article (Illumina Knowledge Article #2843), contact Illumina Technical Support [e-mail].