I can't seem to find a clear answer to this question, so here it goes:

I have sequenced scRNASeq + scVDJSeq (TCR) data, which has been sequenced using feature barcoding from 10x genomics, via antibody capture.

There is a very useful cellranger multi command, which allows us to process both this data types in 'one go'.

In order to run it one needs to generate a csv file a priori, such that cellranger is able to identify which files are pertinent to which file type - see this link:https://support.10xgenomics.com/single-cell-vdj/software/pipelines/latest/using/multi

In the link above, with the example given under "Example multi config CSVs", it seems as though there are separate fastq files for the feature barcode, versus for the actual gene expression samples. Whereas, in my case, I only have the standard R1 and R2 fastq files, which is creating some confusion as how to generate this file, and run cellranger.

Now my question is, how does one run cellranger multi with feature barcoding should there only be the standard 2 fastq files? My understanding is that the feature barcode is usually in the R2 read? Is this correct? Would I need to Demultiplex? I am completely lost as to how to proceed. If you know, could you please provide a short example of what your csv files would look like, should you not have separate fastq files for the feature barcode files?


  • $\begingroup$ How did you create your fastq files? If you've run cellranger mkfastq with the correct sample sheet, you should have got separate fastq files for each library: RNA-seq, VDJ-seq and feature barcode. $\endgroup$
    – Cloudberry
    Mar 27, 2023 at 18:51
  • $\begingroup$ The fastq files were provided by the sequencing company.. We have 4 fastq files (R1,R2,I1,I2) for the GEX & VDJ sequencing, all run via 10x genomics. i.e. I don't have any bcl files, just the fastq's + aligned BAM's $\endgroup$
    – h3ab74
    Mar 27, 2023 at 19:30
  • $\begingroup$ Before running cellranger multi you first need to demultiplex your fastq files to obtain separate files for each library. Unfortunately I've never demultiplexed fastq files so can't recommend any software. $\endgroup$
    – Cloudberry
    Mar 28, 2023 at 20:27

1 Answer 1


Check your FASTQ headers - if they've been demultiplexed, the index barcodes can be found in the header. The barcodes are usually separated by a + in the comment part (second segment when the header is split by " " blank space) of the header. If you see a match to [ATGC]{10}[+][ATGC]{10} in the header, the first [ATGC]{10} is the I1 barcode and the second is the I2 barcode. You can use FFFFFFFFFF placeholder qual scores to generate "fake" I1 and I2 files and then use that with cellranger.

  • $\begingroup$ Thanks for your response. I'm not referring to the I1 & I2 files. I'm referring to feature barcoding technology using antibody capture (or potentially CRISPR). In the examples on the 10x website, they have separate fastq files for these, along with the gene expression fastq's $\endgroup$
    – h3ab74
    Mar 27, 2023 at 18:00
  • $\begingroup$ I see, I apologize for the misunderstanding - I do not know how to help you with feature barcoding. $\endgroup$
    – Ram RS
    Mar 27, 2023 at 18:25

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