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-index
option. 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.
Demultiplexing dual indices with only one index:
In the case with dual indices where the sequencing quality of one index may be lower than anticipated, the other index can sometimes be used for demultiplexing to recover the data. It is recommended to use the --barcode-mismatches argument set to the maximum 2 allowable mismatches to minimize the effect of sequencing errors on demultiplexing.
Run Cell Ranger mkfastq to demultiplex using only the i7 index with 2 allowed mismatches:
cellranger mkfastq --id=FASTQ_output_directory \
--use-bases-mask=Y28n*,I10,N10,Y90n* \
--filter-single-index \
--barcode-mismatches=2 \
--run=/path/to/run/directory/ \
--csv=samplesheet.csv
Make sure that the samplesheet.csv file contains the nucleotide sequences for only the first index:
Lane,Sample,Index
*,sample1,ATGGCTTGTG
*,sample2,CCTTCTAGAG
Run Cell Ranger mkfastq to demultiplex using only the i5 index with 2 allowed mismatches:
cellranger mkfastq --id=FASTQ_output_directory \
--use-bases-mask=Y28n*,N10,I10,Y90n* \
--filter-single-index \
--barcode-mismatches=2 \
--run=/path/to/run/directory/ \
--csv=samplesheet.csv
Make sure that the samplesheet.csv file contains the nucleotide sequences for only the second index (make sure to include the reverse complement if working with data from workflow_b sequencers):
Lane,Sample,Index
*,sample1,CACAACATTC
*,sample2,TCGTTGTATT
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
Related articles:
How to use masking parameter while demultiplexing 10x sequencing data
How can I demultiplex my data if I sequenced 8bp of the index reads instead of 10bp?