# Custom recipes for Illumina Stranded libraries on NextSeq 1000/2000

**Background**\
Read 1 and 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). This "T" makes the first cycle of each read low diversity, which can cause cycle 1 (C1) or fiducial errors.

While this can be mitigated by adding PhiX (5% is recommended), Illumina also provides custom recipes that make this first cycle dark to mitigate this. A dark sequencing cycle will perform chemistry only and omit the imaging step of sequencing by synthesis chemistry.

Please note there is a "loss" of 2 cycles from the NextSeq 1000/2000 sequencing by synthesis (SBS) kit for these dark cycles, as no data is generated during dark cycles but reagents are still used.

**Sequencing Run Setup**

In **NextSeq 1000/2000 Control Software v1.3+**, these custom recipes are **built into the control software**. These recipes can be found under the file path:

/usr/local/illumina/recipes/\[ILMNStrandedTotalRNA **or** ILMNStrandedmRNA]/\[LO **or** MO **or** HO or XO]/\[v3 **-** v7]/recipe.xml

**Table 1**: Sequencing Recipes available with Dark Cycles for Illumina Stranded mRNA or Illumina Stranded Total RNA libraries.

|    | **Standard** | **Standard (600 cycle)** | **XLEAP**              | **XLEAP (600 cycle)** |
| -- | ------------ | ------------------------ | ---------------------- | --------------------- |
| P1 | LO v3        | n/a                      | LO v6 (300 Cycle only) | n/a                   |
| P2 | MO v3        | n/a                      | MO v5                  | n/a                   |
| P3 | HO v3        | n/a                      | HO v5                  | n/a                   |
| P4 | n/a          | n/a                      | XO v5                  | n/a                   |

**IMPORTANT NOTE**: The P1 XLEAP 100 cycle kit and all 600 cycle kits do not have Dark Cycle recipes and will require minimum 5% PhiX spike in.

When selecting an Illumina Stranded library prep kit in sample sheet or in cloud mode, the NextSeq 1000/2000 control software automatically pulls the correct custom recipe; however, it does **not** display that it is using the custom recipe. For the recipe to be pulled automatically, the sample sheet "LibraryPrepKits" field must say "ILMNStrandedTotalRNA" or "ILMNStrandedmRNA" only.

**Run Setup in Standalone mode**

Custom Recipes can also be selected when initiating run in standalone mode on instrument. For instructions, see the "Initiating a Standalone Run" section in the [NextSeq 1000/2000 System Guide](https://support.illumina.com/downloads/nextseq-1000-2000-product-documentation-200027171.html). As previously stated, 2 cycles of SBS will be lost from the total number of cycles to account for the dark cycles.

![](https://761066130-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FGM9W2DuBTgEXv1ClCm8H%2Fuploads%2Fgit-blob-79be804ae517f7c2e27cb470b4f59e1df958cbb7%2Fimage1.png?alt=media\&token=e8a70482-3fc4-44e3-9f98-10e4b1b166d0)

If additional assistance is needed, contact [Illumina Technical Support.](https://www.illumina.com/company/contact-us.html#/united-states/technical-support)

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