10x Cell Ranger Multi

Question

[zizigolu]

enter link description hereHi

I am writing regarding the Cell Ranger Multi analysis for our 10x Genomics 3′ Gene Expression + Cell Multiplexing Capture (CellPlex) libraries.

I get this permanent error

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

This is how my multi config file and feature reference looks like

cat multi_config.csv
[gene-expression]
reference,/users/scratch/cellranger/GRCh38_full/GRCh38_full
cmo-set,/users/scratch/cmo_reference.csv
chemistry,SC3Pv3
create-bam,false

[libraries]
fastq_id,fastqs,feature_types
HB216_gene_exp_C4-AK39983-AK40037_233V53LT3,/users/scratch/fastq_files/X204SC25096963-Z01-F001_01/01.RawData/HB216_gene_exp_C4,Gene Expression
HB216_multip_C4-SCI7T028-SCI5T028_233V53LT3,/users/scratch/fastq_files/X204SC25096963-Z01-F001_01/01.RawData/HB216_multip_C4,Multiplexing Capture

[samples]
sample_id,cmo_ids
HB216_T1,CMO301
HB216_T2,CMO302
HB216_T3,CMO303
HB216_T4,CMO304
HB216_T5,CMO305
HB216_T6,CMO306
HB216_T7,CMO307
HB216_T8,CMO308
HB216_T9,CMO309


cat cmo_reference.csv
id,name,read,pattern,sequence,feature_type
CMO301,CMO301,R2,5P(BC),ATGAGGAATTCCTGC,Multiplexing Capture
CMO302,CMO302,R2,5P(BC),CATGCCAATAGAGCG,Multiplexing Capture
CMO303,CMO303,R2,5P(BC),CCGTCGTCCAAGCAT,Multiplexing Capture
CMO304,CMO304,R2,5P(BC),AACGTTAATCACTCA,Multiplexing Capture
CMO305,CMO305,R2,5P(BC),CGCGATATGGTCGGA,Multiplexing Capture
CMO306,CMO306,R2,5P(BC),AAGATGAGGTCTGTG,Multiplexing Capture
CMO307,CMO307,R2,5P(BC),AAGCTCGTTGGAAGA,Multiplexing Capture
CMO308,CMO308,R2,5P(BC),CGGATTCCACATCAT,Multiplexing Capture
CMO309,CMO309,R2,5P(BC),GTTGATCTATAACAG,Multiplexing Capture

I have downloaded CMO file from 10x website https://www.10xgenomics.com/datasets/10-k-1-1-mixture-of-raji-and-jurkat-cells-multiplexed-2-cm-os-3-1-standard-6-0-0

and this is my code

#!/bin/bash
#SBATCH -J hb16
#SBATCH -p shared
#SBATCH -c 4
#SBATCH --mem=16G
#SBATCH -t 12:00:00
#SBATCH -o hb16.out
#SBATCH -e hb16.err

set -euo pipefail

SCR=~/scratch
CR=${SCR}/cellranger1/cellranger-9.0.1/cellranger
CSV=${SCR}/hb16/HB216_multi_config.csv
OUTDIR=${SCR}/hb16

"${CR}" multi \
  --id=multi_HB216_GRCh38 \
  --csv="$CSV" \
  --localcores=${SLURM_CPUS_PER_TASK} \
  --localmem=$((SLURM_MEM_PER_NODE / 1024))

Please help me to figure out why I get this error

Thanks a lot

[ATpoint]

Looking at the examples https://www.10xgenomics.com/support/software/cell-ranger/latest/analysis/running-pipelines/cr-3p-multi#hashing -- I would assume you need the cmo-set,/users/scratch/cmo_reference.csv line under [feature], not under [gene-expression]?

[zizigolu]

Thanks a lot

But in this section

cmo-set Optional. The default CMO reference IDs are built into the Cell Ranger software and do not need to be specified. However, this option can be used to specify the path to a custom CMO set CSV file, declaring CMO constructs and associated barcodes. See CMO Reference section for details.

In this thread

https://www.10xgenomics.com/support/software/cell-ranger/7.2/advanced/cr-multi-config-csv-opts

cmo comes in gene expression section :( But the way I get the same error