> For the complete documentation index, see [llms.txt](https://knowledge.illumina.com/llms.txt). Markdown versions of documentation pages are available by appending `.md` to page URLs; this page is available as [Markdown](https://knowledge.illumina.com/library-preparation/general/library-preparation-general-reference_material-list/000003708.md).

# 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 a monotemplate 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 call 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.

See the following sections for instrument specific guidance for sequencing Illumina Stranded RNA libraries.

**MiSeq i100**

Improvements to the MiSeq i100 Series base calling software as well as the flow cell architecture allow this platform to sequence Illumina Stranded RNA libraries without requiring Dark Cycle recipes to skip this first cycle. Illumina recommends adding **at least 2% PhiX** spike in when running Illumina Stranded mRNA Prep, Ligation or Illumina Stranded Total RNA Prep Ligation with Ribo-Zero Plus libraries as a positive control.

Additionally, override cycles should be used to mask the first cycle in the FASTQ when using any of the onboard or Cloud DRAGEN analysis workflows.

See [How to use the 'OverrideCycles' setting in the Run Planning User Interface](/software/general/software-general-faq-list/000005671.md) for assistance with configuring override cycles.

**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](/instrumentation/nextseq-1000-2000/instrumentation-nextseq-1000-2000-faq-list/000005583.md).

**NovaSeq 6000**

As there is no dark cycle recipe for Illumina Stranded RNA libraries on the NovaSeq 6000, it is recommended 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 or later. 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](/instrumentation/novaseq-x-x-plus/instrumentation-novaseq-x-x-plus-reference_material-list/000008394.md).

**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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