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.

MiSeq i100

As there is no dark cycle recipe for Illumina Stranded RNA libraries on the MiSeq i100, it is important to spike in at least 2% PhiX for Illumina Stranded RNA libraries sequenced on the MiSeq i100.

NextSeq 1000/2000

If not using a dark cycle recipe, Illumina recommends 5% PhiX for Illumina Stranded 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 Stranded 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 5% PhiX for Illumina Stranded RNA libraries sequenced on the NovaSeq X/X Plus, when on control software v1.3. For control software v1.2 or lower, 10-15% PhiX is recommended.

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].