Custom recipes for Illumina Stranded libraries on NextSeq 1000/2000
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.
Custom recipes can be found on the following Illumina Support webpages:
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]/[v1 or v2 or v3]/recipe.xml
Note: not all versions have all outputs
When selecting an Illumina Stranded library prep kit in sample sheet or in cloud mode, the NextSeq 1000/2000 control software will automatically pull the correct custom recipe; however, it will 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".
For previous software versions, add the custom recipes linked above to the relevant recipe folder(s), then select the custom recipe during run set up, as shown below. The default recipe folder is: /usr/local/illumina/recipes
For additional information, contact Illumina Technical Support.
As previously stated, 2 cycles of SBS will be lost from the total number of cycles to account for the dark cycles.
Standalone mode Custom Recipes can also be selected when setting up the instrument in standalone mode. For instructions, see the “Initiating a Standalone Run” section in the NextSeq 1000/2000 System Guide.
For any feedback or questions regarding this article (Illumina Knowledge Article #5583), contact Illumina Technical Support techsupport@illumina.com. |
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