Question: How can I demultiplex a single-indexed library that was sequenced on a dual-indexed flow cell?
Answer: How you go about demultiplexing single-indexed libraries on dual-indexed flow cells depends on which Cell Ranger version you are working with.
Cell Ranger 5.0 and later:
If you have single-indexed libraries that were sequenced on a dual-indexed flow cell, you can still use mkfastq to generate fastqs. You will need to run the mkfastq command as usual but also specify the --filter-single-indexoption. Additionally, you need to mask the second index with --use-bases-mask, for example: --use-bases-mask=Y28n*,I8n*,N10,Y90n*. Please also see Generating FASTQs with cellranger mkfastq for details.
Note that if you have single- and dual-indexed libraries sequenced on the same dual-indexed flow cell, you'll need to demultiplex the two types of libraries separately. For example, you can first demultiplex single-indexed libraries with --filter-single-index and the --use-bases-mask options as long as the input sample sheet contains only single-indexed sample information. You can then demultiplex dual-indexed libraries in a separate run by specifying the --filter-dual-index option with input sample sheet containing only dual-indexed sample information.
Cell Ranger 4.0:
You will need to run bcl2fastq directly as demultiplexing single-indexed libraries in dual-indexed flow cells is not supported in mkfastq for this Cell Ranger version. Note that mkfastq still works for single-indexed libraries sequenced on single-indexed flow cells.
Cell Ranger 3.1 or earlier:
If you have single-indexed libraries that were sequenced on a dual-indexed flow cell 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: --use-bases-mask=Y28n*,I8n*,N10,Y90n*.
Single Cell ATAC libraries, by contrast, are required to be dual-indexed. Please see Sequencing Requirements for Single Cell ATAC.
Products: Single Cell Gene Expression
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