Single tile drop outs affecting metrics on the NextSeq 1000/2000

In some NextSeq 1000/2000 runs, it is possible to see a single tile or a small number of tiles in which the quality drops. If this occurs within the first 25 cycles, the passing filter can be negatively impacted within the tile(s). If this occurs at a later point in the run, it is possible to see small shifts in FWHM (the average full width of clusters at half maximum (in pixels)) or intensity.

Cause: The most common cause for this is a small bubble or bubbles present in the flow cell. Bubbles are more likely to affect the bottom surface but can sometimes impact both the top and bottom surface. It is most likely to see this with a small number of consecutive cycles at the beginning of the run.

Bubbles may result from improper reagent handling. Make sure each reagent cartridge is prepared for sequencing following recommended guidelines, see NextSeq 1000/2000 Product Documentation Thaw the Bagged Cartridge. For any runs impacted with single tile dropout, review the run as a whole for meeting output specifications (Specifications for the NextSeq 1000/2000). Single tile dropouts are very unlikely to cause a run to fail to meet specifications. If run does not meet yield or Q30 specifications, evaluate for other potential causes.

Identification: In this example, the drop out occurred during the first 25 cycles which caused the tile to not pass filter.

(Dark blue tiles can indicate much lower or zero values compared to other tiles.)

Effects of the bubble can be observed as an intensity drop out for a few cycles, followed by intensity recovering.

This is an additional example of a single tile drop-out occurring later in the run. This example presented as small FWHM spikes.

FWHM for the given tile at cycle 72 higher than surrounding tiles, recovers in the subsequent cycle.

Q30 was not calculated for the impacted tile.

For any feedback or questions regarding this article (Illumina Knowledge Article #2723), contact Illumina Technical Support [e-mail].