PhiX requirements for sequencing Illumina Stranded RNA libraries

Background

Insert Read 1 and Insert Read 2 of Illumina Stranded mRNA Prep, Ligation and Illumina Stranded Total RNA Prep Ligation with Ribo-Zero Plus/Ribo-Zero Plus Microbiome libraries begin with 100% T-base because of how the adapters are added. This 100% T base makes the first cycle of each insert read low in base diversity, which can cause cycle 1 (C1) or fiducial registration errors if not mitigated with a dark cycle sequencing recipe or sufficient PhiX spike in.

NextSeq 1000/2000

If not using a dark cycle recipe, Illumina recommends 5% PhiX for Illumina RNA libraries being sequenced on the NextSeq 1000/2000.

For more information on dark cycle recipes on NextSeq 1000/2000, see Custom recipes for Illumina Stranded libraries on NextSeq 1000/2000

NovaSeq 6000

As there is no dark cycle recipe for Illumina RNA libraries on the NovaSeq 6000, it is important to spike in at least 5% PhiX for Illumina Stranded RNA libraries sequenced on the NovaSeq 6000.

NovaSeq X Series

If not using a dark cycle recipe, Illumina recommends at least 10% PhiX for Illumina Stranded RNA libraries sequenced on the NovaSeq X/X Plus.

For more information on dark cycle recipes on the NovaSeq X Series, see Custom recipes for Illumina Stranded libraries on NovaSeq X Series

Other Illumina sequencers

For sequencing instruments not specifically listed above, there are no dark cycle recipe options. For these instruments, Illumina recommends spiking in 5-10% PhiX.

For any feedback or questions regarding this article (Illumina Knowledge Article #3708), contact Illumina Technical Support [e-mail].