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. 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. As previously stated, 2 cycles of SBS will be lost from the total number of cycles to account for the dark cycles.

If additional assistance is needed, contact Illumina Technical Support.

For any feedback or questions regarding this article (Illumina Knowledge Article #5583), contact Illumina Technical Support [e-mail].