Question: I have multiplexed dual and single indexed libraries on the same lane of an Illumina flow cell. How can I generate fastqs for both library types ?
Answer: If you have sequenced single and dual indexed libraries together on the same lane of a flowcell, you will need to use Cell Ranger 4.0 or later. And you will also need to run mkfastq twice: once for single indexed libraries and a second time for dual indexed libraries. For dual indexed samples, the sample sheet must match both the i7 and i5 indices, whereas for the single-indexed samples, only the i7 index is used.
For demultiplexing only dual-indices, use the option --filter-dual-index argument. This argument processes samples identified by i7/i5 dual-indices (e.g., SI-TT-A6), ignoring single-index samples.
For demultiplexing only single-indices, in a dual-indexed flowcell, you will need to run bcl2fastq directly if you are working with Cell Ranger 4.0. Please see this article for more details.