PhiX Spike In Requirements for Low Diversity Libraries on NovaSeq X Series Instruments

Background

When sequencing libraries with low base diversity on the NovaSeq X Series, unbalanced nucleotide composition can negatively impact template registration along with data quality and data output. 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.

PhiX Spike-In Recommendations for NovaSeq X Series

The table below shows the percent PhiX spike-in and recommended library loading concentrations for various libraries on NovaSeq X Series instruments. Typically, low diversity libraries on the NovaSeq X Series require a 10-20% PhiX spike-in. For third party or custom libraries that are not optimized on the NovaSeq X Series, PhiX spike-in titration experiments should be performed alongside loading concentration optimization to achieve the best performance.

PhiX titration on Illumina 16S libraries

When testing 16S libraries prepared using the Illumina 16S Library Preparation protocol on the NovaSeq X Series, titrations were performed spiking in 1%, 10%, 20%, and 40% PhiX, and assessing %Pass Filter (%PF) and %Occupancy (Figure 1), Quality in Read1 and Read2 (Figure 2), and %Aligned (Figure 3).

Figure 1: Relationship of %PF and %Occupied at various PhiX spike in percentages for 16S libraries sequenced on NovaSeq X Series instruments.

Figure 2: Percentage of bases with Q30 or greater in Read 1 and Read 2 at increasing PhiX spike-in percentages.

Figure 3: Relationship of %Aligned to PhiX spike-in when sequencing 16S libraries on the NovaSeq X Series instruments.

These results indicate that at least 20% PhiX spike-in is required to maximize the %PF, % Occupancy, and quality of Read1/2. Maximizing these factors ensures robust base calling and reliable secondary analysis on NovaSeq X Series for Illumina 16S libraries.

Also of note in this experiment, 20% PhiX spike-in results in around 20% PhiX alignment. Library size and other intrinsic library characteristics can result in the PhiX %Aligned not matching the spike-in rate. PhiX spike-in amounts should be titrated for each library type to ensure sufficient PhiX Alignment for the best results.

PhiX titration on ZymoBIOMICS 16S Microbial Standard libraries

When testing ZymoBIOMICS 16S libraries on the NovaSeq X Series, titrations were performed spiking in 1%, 10%, 20%, and 40% PhiX, and assessing genus level classification. A total of 8 bacterial species are expected based on the input sample type.

The results of this experiment indicate that at least 10% PhiX spike-in is required for robust classification at the genus level when running ZymoBIOMICS 16S libraries on the NovaSeq X Series. Insufficient PhiX resulted in high levels of unclassified reads and reduced detection of the 8 expected species.

For more information about the PhiX control and % PhiX requirements on other sequencing platforms, refer to the article How much PhiX spike in is recommended when sequencing low diversity libraries on Illumina platforms?

For any feedback or questions regarding this article (Illumina Knowledge Article #8913), contact Illumina Technical Support [e-mail].