Custom recipes for Illumina Stranded libraries on NovaSeq X 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. This "T" makes the first cycle of each read low diversity, which can cause reduced Pass Filter (PF) clusters and quality.

Illumina provides a dark cycle recipe that skips imaging step of this cycle for both Read 1 and Read 2. A dark sequencing cycle will perform chemistry only and omit the imaging step of sequencing by synthesis (SBS) chemistry. Note, there is a "loss" of 2 cycles from the NovaSeq X/X Plus SBS kit for these dark cycles, as reagents are still used, though no data is generated during dark cycles. This 2 cycle loss applies to all lanes in the NovaSeq X Series run if the recipe is invoked.

Sequencing Illumina Stranded RNA Libraries in all lanes on NovaSeq X SeriesWhen selecting either Illumina Stranded RNA library prep kit in the Run Planning tool on instrument or in BaseSpace, the software automatically selects the correct dark cycle recipe, provided that this is the only library type sequenced on the entire flow cell. However, it will not display that it is using the dark cycle recipe. The addition of any other library type to any other lane through additional configurations will result in the default recipe being applied.

Local runs with analysis started with V2 sample sheets will pull the dark cycle recipe if the "LibraryPrepKits" line contains the value "ILMNStrandedmRNA" and/or "ILMNStrandedTotalRNA." Note that the "ILMNStrandedmRNA" recipe is used by both Illumina Stranded mRNA and Illumina Stranded Total RNA libraries. If the LibraryPrepKits value is one/both of these and another library prep kit, the default recipe is applied.

Sequencing Illumina Stranded RNA Libraries + Other Libraries on NovaSeq X SeriesBecause the dark cycle recipe applies to all lanes of the NovaSeq X instrument, other library types may be negatively impacted in mixed library runs where this recipe is applied. The following table indicates which libraries are compatible with the dark cycle recipe required for Illumina Stranded RNA libraries:

Mixed Library Runs Using the Dark Cycle Recipe

For mixed library runs that are compatible with the Illumina Stranded RNA dark cycle recipe, the dark cycle recipe must be manually applied to the run. To manually apply the dark cycle recipe to a run set up via any mode, including manual runs, select the file path of the dark cycle recipe during run setup:

On NovaSeq X Series instruments, this dark cycle recipe is built into the control software, on Software Suite v1.2 and higher.

For 1.5B flow cells, this recipe can be found under the file path: /usr/local/illumina/consumables/ILMNStrandedmRNA/1/recipe.xml

For 10B flow cells, this recipe can be found under the file path: /usr/local/illumina/consumables/ILMNStrandedmRNA/3/recipe.xml

For 25B flow cells (requires control software v1.2), this recipe can be found under the file path: /usr/local/illumina/consumables/ILMNStrandedmRNA/v2/100/recipe.xml /usr/local/illumina/consumables/ILMNStrandedmRNA/v2/200/recipe.xml /usr/local/illumina/consumables/ILMNStrandedmRNA/v2/300/recipe.xml

As previously stated, 2 SBS cycles are lost from the total number of cycles to account for the dark cycles. This can be compensated by increasing the number of cycles in each Read from 151 to 152, provided the total number of cycles in the cartridge are not exceeded.

Mixed Library Runs Not Using the Dark Cycle Recipe

For mixed library runs where the other libraries are not compatible with the Illumina Stranded RNA dark cycle recipe, use the default recipe for sequencing. The default recipe will be automatically selected if any additional library types are added to the run configuration. Lanes containing Illumina Stranded RNA libraries should have at least 5% PhiX spiked-in to mitigate the low diversity caused by the first T base, when on control software v1.3. On control software v1.2 or lower, 10-15% PhiX spike-in is recommended.

For additional information, contact Illumina Technical Support.

For any feedback or questions regarding this article (Illumina Knowledge Article #8394), contact Illumina Technical Support [e-mail].