Troubleshooting elevated PhiX alignment in sequencing runs

The PhiX Control v3 Library (commonly referred to as PhiX, FC-110-3001) is commonly added to sequencing runs as a positive control for sequencing performance or to help balance low-diversity libraries. The addition of PhiX to a sequencing run also allows for additional sequencing metrics to be calculated during a run, enabling deeper analysis of loading optimization when comparing the %Align metric to the expected PhiX Spike-in by volume.

Occasionally, users may find that the %Align metric reports a significantly higher value than was spiked-in. Unlike low PhiX alignment which can have multiple root causes, high PhiX alignment is the result of PhiX taking up a larger than expected proportion of the sequenced material on a run. There are several possible causes for this phenomenon.

1. Inaccurate quantification of the library and/or PhiX.

2. Too low of a loading concentration used for the library pool.

3. Issue with library design resulting in poor clustering or priming.

Sequencer or sequencing consumable performance issues impact libraries and PhiX alike, so elevated PhiX alignment is not indicative of instrument malperformance or poor reagent quality.

Interpreting sequencing results to determine causes of high PhiX alignment

Users can determine which of the possible causes are resulting in high PhiX by examining the sequencing metrics of their run. If a run has high Q30 scores, low error rates, and either low density (if sequencing with a non-patterned flow cell like MiSeq or NextSeq 500/550 systems) or low %Occupancy (if sequencing with a patterned flow cell like iSeq 100, MiSeq i100, NextSeq 1000/2000, NovaSeq 6000, or NovaSeq X/X Plus systems), this indicates that the run is under-clustered. The root cause in this case is an error in quantification, QC, too low of a library loading concentration, or design which resulted in PhiX taking up a larger proportion of the sequenceable material on the flow cell than expected.

On the other hand, if a run shows lower Q30 scores, high density/occupancy, and higher error rate along with higher than expected PhiX Alignment, this could be an indication of run over-clustering in which PhiX was loaded at a higher concentration than expected and resulted in elevated clustering. Users may also see these phenotypes in combination with low Index 1 quality or a high proportion of Undetermined reads after FASTQ generation, both of which may be a sign of high PhiX presence in a sequencing run.

If a run is indicating >90% PhiX Alignment, this is the result of a total or near-total clustering failure of the library and is related to library design or compatibility issues.

Troubleshooting high PhiX alignment

1. Quantify both PhiX and the library pool to verify accurate loading concentration calculations. Illumina recommendations for quantification methods can be found in the Knowledge article Library quantification and quality control quick reference guide

   1. If the PhiX stock concentration is significantly higher than the expected 10 nM, contact Illumina Technical Support. The PhiX can still be used on future runs, but loading calculations should be adjusted to reflect the measured concentration of PhiX.

   2. If library pool concentrations are significantly lower than expected, adjust dilution concentrations to achieve optimal loading for the sequencer used. Reference the Denature and Dilution Guide for Illumina Library preparation methods.

   3. If quantification is as expected, it may be necessary to use a higher loading concentration for the library pool for optimal clustering.

2. In cases where PhiX Alignment is >90%, libraries should be remade to make sure all required regions of the library adapters are present and that the libraries are able to successfully bind to the flow cell. Make sure that libraries are compatible with Illumina instruments, especially when using custom primers and/or third party kits. More information can be found in the Knowledge article Considerations when migrating non-Illumina libraries between sequencing platforms.

For assistance with any troubleshooting steps, contact Illumina Technical Support.

For any feedback or questions regarding this article (Illumina Knowledge Article #9236), contact Illumina Technical Support [email protected].