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Custom recipes for Illumina Stranded libraries on NovaSeq X
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.
While this can be mitigated on the NovaSeq X/X Plus by spiking in at least 10% PhiX, Illumina also provides a dark cycle recipe that essentially skips 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 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/X Plus run if the recipe is used.
On NovaSeq X and NovaSeq X Plus, this dark cycle recipe is built into the control software. 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/4/recipe.xml
When selecting either Illumina Stranded RNA library prep kit in the Run Planning
tool on instrument or in BaseSpace, the software will automatically select 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" 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 of these and another library prep kit, the default recipe is applied.
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:
As previously stated, 2 SBS cycles will be 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.
For any feedback or questions regarding this article (Illumina Knowledge Article #8394), contact Illumina Technical Support [email protected]. |
Last modified 18d ago