Question: I have a dual-indexed library. However, I sequenced it with an 8bp single index (i7) configuration. Will sequencing only the first 8bp of the i7 index (without the i5 index) be sufficient for demultiplexing?
Answer: Yes, it is possible to demultiplex the library when only the first 8bp of the i7 index were sequenced. This applies to Single Cell 3' Gene Expression, Single Cell 5' Immune Profiling, and Visium Spatial Gene Expression.
The 10x dual index plates are designed such that the index sequence will be unique even if you sequence only the first 8bp of the i7 index. However, since you will be demultiplexing using only a single index, you will lose the benefit of dual indexing in removing potential index hopped reads.
To demultiplex the data, you will need to specify the first 8 bases of each sample index in the sample sheet. The sample indices can be found on the support site:
- 3' Gene Expression (and Feature Barcode) sample indices
- 5' Immune Profiling (and Feature Barcode) sample indices
- Visium sample indices
Suppose you used the index SI-TT-A1, which consist of sequences as below:
"SI-TT-A1": {
"index": "GTAACATGCG",
"index2_workflow_a": "AGTGTTACCT",
"index2_workflow_b": "AGGTAACACT"
Running mkfastq:
You can still run mkfastq pipeline with simple CSV sample sheet. The index sequence needs to be specified as below, and you cannot use the index name (SI-TT-A1) in the sample sheet.
Lane,Sample,Index *,My_sample,GTAACATG
Running bcl2fastq:
Using bcl2fastq directly for demultiplexing is also an option. Please see corresponding support pages for instructions:
- bcl2fastq for 3' Gene Expression (and Feature Barcode) libraries
- bcl2fastq for 5' Immune Profiling (and Feature Barcode) libraries
- bcl2fastq for Visium libraries
Note: The information above does not apply to any ATAC libraries. For single assay ATAC or multiome ATAC sequencing, it is mandatory to have 8 bp on i7 reads for demultiplexing, and 16bp on i5 reads for the cellular barcodes.
Products: Single Cell Gene Expression, Single Cell Immune Profiling, Visium Spatial Gene Expression
Related Article: How can I demultiplex my data if I sequenced 8bp of the index reads instead of 10bp?