Question: Should I select Workflow A or Workflow B for the i5 index sequence?
Answer: When sequencing the i5 index, Illumina instruments can use two different workflows:
- Workflow A = forward strand workflow
- Workflow B = reverse complement workflow
To determine whether your Illumina instrument uses the forward or reverse complement workflow, please refer to Illumina's Indexed Sequencing Overview Guide:
-
The forward strand workflow is performed on the:
- NovaSeq 6000 with v1.0 reagent kits
- MiSeq
- MiniSeq with Rapid Reagent kits
- HiSeq 2500, HiSeq 2000
-
The reverse complement workflow is performed on the:
- NovaSeq 6000 with v1.5 reagent kits
- iSeq 100
- MiniSeq with Standard reagent kits
- NextSeq Systems
- HiSeq X, HiSeq 4000, HiSeq 3000
Whether you use the forward or reverse orientation of the i5 index will also depend on the software that is being used. Please see the following article from Illumina for further guidance:
Guidelines for reverse complementing i5 sequences for demultiplexing
In the CSV files for 10x Genomics Dual Index plates, both the Workflow A and Workflow B sequences are listed for the i5 sequence. Note that the Workflow B sequence is the reverse complement of the Workflow A sequence.
Products: Single Cell Gene Expression, Single Cell Immune Profiling, Single Cell Gene Expression Flex, Single Cell Multiome ATAC + Gene Expression, Single Cell ATAC, Visium for Fresh Frozen, Visium for FFPE, Visium CytAssist Spatial Gene and Protein, Visium CytAssist for FFPE