Cellranger: Demux pooled (hashing antibodies) GEX and VDJ 10x sequence fastq data

Question

[Flipstone]

Situation... 3 individuals are pooled, the pbmcs for these individuals are incubated with hashing antibodies prior to sorting. For these individuals 5' GEX and VDJ 10x sequencing has been performed.

The results are GEX and VDJ data for these pooled samples for which I have the fastqs as follows:

GEX

SAMPLEGEX_*_L001_R1_001.fastq.gz
SAMPLEGEX_*_L001_R2_001.fastq.gz

VDJ:

SAMPLEVDJ_*_L001_R1_001.fastq.gz
SAMPLEVDJ_*_L001_R2_001.fastq.gz

And also the I1 and I2 fastqs ( and then again the same for L002).

This is all data I currently have, so both GEX and VDJ data are pooled...

I tried to follow this guide:

https://www.10xgenomics.com/analysis-guides/demultiplexing-and-analyzing-5%E2%80%99-immune-profiling-libraries-pooled-with-hashtags

However, I need to specify GEX fastqs as well as Multiplexing Capture fastqs? I only have GEX (and VDJ).

I then modifed the GEX fastqs as described here:

https://kb.10xgenomics.com/hc/en-us/articles/4407386498957-I-used-antibody-tags-for-cell-surface-protein-capture-and-cell-hashing-with-Single-Cell-3-chemistry-How-can-I-use-Cell-Ranger-to-analyze-my-data

In order to use these as the fastqs for multicapture/cell multiplexing...

For this I created the following 'hashing_demux-set.csv' specifying which hashing antiobody (sequences) were used:

id,name,read,pattern,sequence,feature_type
Hash-tag1,Hash-tag1,R2,^NNNNNNNNNN(BC)NNNNNNNNN,GTCAACTCTTTAGCG,Multiplexing Capture
Hash-tag2,Hash-tag2,R2,^NNNNNNNNNN(BC)NNNNNNNNN,TGATGGCCTATTGGG,Multiplexing Capture
Hash-tag3,Hash-tag3,R2,^NNNNNNNNNN(BC)NNNNNNNNN,TTCCGCCTCTCTTTG,Multiplexing Capture

And the following 'demux_config.csv':

[gene-expression]
reference,/path/to/ref/refdata-gex-GRCh38-2024-A
cmo-set,/path/to/hashing_demux-set.csv
create-bam,true

[libraries]
fastq_id,fastqs,lanes,feature_types
SAMPLEGEX_,/path/to/fastq/org/,1|2,Multiplexing Capture
SAMPLEGEX_,/path/to/fastq/mod/,1|2,Gene Expression

[samples]
sample_id,cmo_ids
sample1,Hash-tag1
sample2,Hash-tag2
sample3,Hash-tag3

Running the cellranger pipeline as follows:

cellranger multi --id=demultiplexed_samples --csv=demux_config.csv --localcores=4

But this results (after hours) in the error:

[error] Deplex Error: No cell multiplexing tag sequences were detected in the Multiplexing Capture library. Common causes include:

Wrong pattern or sequences provided in the feature reference (CMO reference) csv file.

Corrupt or low quality reads.

Incorrect input fastq files for the Multiplexing Capture library. Contact support for additional help with this error.

Can anyone tell me if I understand this completely wrong? And/or what the proper way to approach this would be...

Also, when trying to grep the hash-tag sequences from the fastqs I don't seem to get any results... so I feel like I miss something essential here.

[bk11]

Since you do not have Multiplexing libraries corresponding to your GEX and VDJ libraries, you cannot use CellRanger LMO/CMO based method for demultiplexing your data. If you have genotype data for your subjects, you can use genotype approaches to demultiplex your data. See the approaches here.

[Flipstone]

Hmmm...Thanks for the response, but this is all data I currently have as far as I know... no genotyping data. I will contact the one supplying the data if there is more that was not sent yet.

I did find that link before as well, and was also looking more into cell/nuclear hashing options but so far couldn't really connect the dots, will look more into those as well.