# 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](https://www.illumina.com/content/dam/illumina/gcs/assembled-assets/marketing-literature/illumina-stranded-rna-t-overhang-tech-note-470-2020-010/illumina-stranded-rna-t-overhang-tech-note-470-2020-010.pdf). 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](https://knowledge.illumina.com/instrumentation/nextseq-1000-2000/instrumentation-nextseq-1000-2000-faq-list/000005583)

**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](https://knowledge.illumina.com/instrumentation/novaseq-x-x-plus/instrumentation-novaseq-x-x-plus-faq-list/000008394)

**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.

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| *For any feedback or questions regarding this article (Illumina Knowledge Article #3708), contact Illumina Technical Support* [*techsupport@illumina.com*](mailto:techsupport@illumina.com?subject=Question%2FFeedback%20Regarding%20Illumina%20Knowledge%20Article%20#000003708%20-%20Library%20Preparation%20\&body=Dear%20Illumina%20Technical%20Support,%0D%0A%0D%0A)*.* |


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