# Sequencing Illumina Stranded Total RNA and mRNA Libraries on the MiSeq i100 Series

**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** libraries begin with a "T" 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). On some platforms, this can impact clusters passing filter and base quality for cycle 1. On these platforms, this is avoided by using Dark Cycle recipes that perform a chemistry-only step for the first cycle of each Insert Read, or by adding sufficient PhiX to balance the diversity in the first cycle.

**Guidance for the MiSeq i100 Series**

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 Knowledge article [How to use the 'OverrideCycles' setting in the Run Planning User Interface](https://github.com/illumina-swi/illumina-knowledge/blob/master/articles/Knowledge/How-to-use-the-OverrideCycles-setting-with-BCL-Convert-on-BaseSpace/README.md) for assistance with configuring override cycles.

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| *For any feedback or questions regarding this article (Illumina Knowledge Article #9350), contact Illumina Technical Support* [*techsupport@illumina.com*](mailto:techsupport@illumina.com?subject=Question%2FFeedback%20Regarding%20Illumina%20Knowledge%20Article%20#000005671%20-%20Instrumentation%20\&body=Dear%20Illumina%20Technical%20Support,%0D%0A%0D%0A)*.* |


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