How much PhiX spike in is recommended when sequencing low diversity libraries on Illumina platforms?

When sequencing libraries with low base diversity, unbalanced nucleotide composition can negatively impact cluster mapping (on non-patterned flow cells) and template registration (on non-patterned flow cells) along with data quality and data output.
For more information on the importance of base diversity on Illumina sequencing platforms, refer to the bulletin What is nucleotide diversity and why is it important?
To compensate for low base diversity in libraries, Illumina recommends spiking in the PhiX Control v3 Library (catalog number FC-110-3001, commonly referred to as PhiX) for sequencing. The PhiX Control v3 Library has a diverse base composition (45% GC and 55% AT) that provides the balanced fluorescent signals that low diversity sample libraries lack during each sequencing cycle. This, in turn, assists with cluster mapping/template registration and improves overall run performance.
For more information about using PhiX Control v3 Library, refer to the bulletin What is the PhiX Control v3 Library and what is its function in Illumina Next Generation Sequencing?
This table lists the percentage of PhiX Control v3 library Illumina recommends spiking in when running low diversity libraries on the indicated sequencing platforms and control software versions.
PhiX Aligned (%)†
iSeq 100
Minimum 5%
MiSeq (MCS 2.2 or higher)
Minimum 5%
NextSeq 500/550
NextSeq 1000/2000
HiSeq 1000/1500/2000/2500 (HCS 2.2.38 or higher)
Minimum 5%
HiSeq 3000/4000 (HCS 3.3.76 or lower)
HiSeq 3000/4000 (HCS 3.4.0 or higher)
NovaSeq 6000
Minimum 5%
Differences in clustering efficiency between PhiX and the sample library can affect the PhiX spike-in percentage required to achieve the above-targeted percent PhiX aligned. For example, more PhiX may be required if the sample library clusters more efficiently than PhiX. Contact Technical Support at [email protected] with any questions about your particular library and platform. *PhiX can be further adjusted based on experimentation. Illumina recommends starting with higher spike-in percentages and reducing based on run performance.
** For low diversity libraries using NextSeq 1000/2000 P1 and P2 600 cycle kits, at least 32% of reads should align to PhiX for optimal performance. We recommend to spike in 40% to account for potential variation in final % read alignment metric. Dependent on the type of low diversity library, this may be reduced after optimization.
Frequently Asked Questions:
  • Can the PhiX Control v3 Library provide improved nucleotide diversity for low diversity index sequences?
    • No, the PhiX Control v3 Library is unindexed and does not balance signals ing index reads.
  • Are there other considerations when sequencing low diversity libraries?
For any feedback or questions regarding this article (Illumina Knowledge Article #1527), contact Illumina Technical Support [email protected].
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