Question: How can I demultiplex a single indexed library that was sequenced on a dual-indexed flowcell?
Answer: How you demultiplex single indexed libraries on dual indexed flowcells depends on the Cell Ranger version you are working with.
Cell Ranger 4.0:
You will need to run bcl2fastq directly as demultiplexing single indexed libraries in dual indexed flowcells is not supported in mkfastq of this Cell Ranger version. Note that mkfastq still works for single indexed libraries sequenced on single indexed flowcells.
Cell Ranger 3.1 or earlier:
If you have single indexed libraries that were sequenced on a dual indexed flowcell you can still use mkfastq or bcl2fastq to generate fastqs. You will need to run the
mkfastq command as usual but also specify
--ignore-dual-index option. Otherwise, you will see this error:
[error] Dual-index flowcell detected. Please add the --ignore-dual-index option to proceed, or use an Illumina Experiment Manager-formatted sample sheet with an index2 column for the second index.
Additionally, you need to mask the second index with
--use-bases-mask, for example:
Single Cell ATAC libraries, by contrast, are required to be dual-indexed. Please see Sequencing Requirements for Single Cell ATAC.