How to troubleshoot low percent clusters passing filter (%PF) on the NovaSeq X/X Plus

The NovaSeq X/X Plus uses patterned flow cell technology. Patterned flow cells contain billions of nanowells at fixed locations across both surfaces of the flow cell. The number of nanowells occupied with a single cluster that passes filter is directly proportional to the number of reads passing filter. Lower than expected clusters passing filter, also called % Pass Filter or %PF, can result from a variety of causes including library quality, consumables, or instrument hardware.

For expected reads passing filter specifications, see the NovaSeq X Series Specifications page. While not a specification itself, runs that have %PF greater than 60-65% will typically meet or exceed the output specification and are considered successful.

If a lane or sequencing run shows lower than expected %PF but is above 10%, see the following.

Troubleshooting steps:

1. Check if the sequencing run is under- or over-clustered by plotting the %Occupied versus %PF.

• For details on how to generate these graphs, see Plotting %Occupied by %PF to optimize loading for the NovaSeq X/X Plus, NovaSeq 6000, and iSeq 100.

1. If the run is under-clustered:

   1. Check PhiX %Aligned; if it is higher than the spike in, this often indicates a library quantification or quality issue.

   2. Review and re-quantify library pool concentration to make sure it is accurate.

   3. Consider increasing the library loading concentration.

2. If the run is over-clustered:

   1. Over-clustering cannot be caused by instrument or consumable-related root causes.

   2. Check PhiX %Aligned; if it is lower than the spike in, this can suggest a library quantification issue.

   3. Review and repeat library pool concentration to make sure it is accurate.

   4. Review library traces for short insert or adapter dimer contamination.

   5. Reduce the library loading concentration.

3. If the library pool is low in base diversity, increasing the PhiX spike-in can often increase the %PF by providing additional nucleotide balance.

If the %PF value for the run or a specific lane is less than 10%, this can indicate a potential clustering failure. If this is observed or additional troubleshooting assistance is required, contact Illumina Technical Support.

For any feedback or questions regarding this article (Illumina Knowledge Article #7783), contact Illumina Technical Support [e-mail].