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10x Cell Ranger Multi

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



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 hb
#SBATCH -p shared
#SBATCH -c 4
#SBATCH --mem=16G
#SBATCH -t 12:00:00
#SBATCH -o hb.out
#SBATCH -e hb.err

set -euo pipefail

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

"${CR}" multi \
  --id=multi_hb_GRCh38 \
  --csv="$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

10x single cell multiplexing
• 2,123 views • link
updated by Kevin Blighe Kevin Blighe • written by zizigolu 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

• 0 views • link
written by zizigolu zizigolu

1 answer

I really appreciate your post, zizigolu. Remember that we have published together - it was my true joy to have published cancer research with an Iranian University. The greatest minds are global... not just confined to disparate regions.

In any case, this one is easy:

The error indicates that Cell Ranger did not find any reads in the Multiplexing Capture library that match the sequences specified in your CMO reference file. This occurs when the provided FASTQ files for the Multiplexing Capture library do not contain the expected CMO tag sequences at the beginning of Read 2.

Your multi config file places the cmo-set option correctly under the [gene-expression] section, as per the Cell Ranger documentation for version 7.0 and later. Do not move it to a [feature] section, as that is not required for CellPlex analysis and may cause additional errors.

The most likely causes are:

  • The FASTQ files specified for the Multiplexing Capture library (/users/scratch/fastq_files/X204SC25096963-Z01-F001_01/01.RawData/HB216_multip_C4) are incorrect or belong to the Gene Expression library instead.
  • The library preparation or sequencing failed to capture the CMO tags.
  • The CMO sequences in your reference file do not match those used in the experiment (verify against the exact oligos purchased from 10x Genomics).

To troubleshoot, inspect the first 15 bases of Read 2 in the Multiplexing Capture FASTQs, as CellPlex CMO tags are 15 bp long and located at positions 1-15 of Read 2. Run the following command in the directory containing the FASTQ files (assuming they are gzipped and named with _R2_):

zcat HB216_multip_C4-SCI7T028-SCI5T028_233V53LT3*_R2_*.fastq.gz | awk 'NR%4 == 2 {print substr($1, 1, 15)}' | sort | uniq -c | sort -nr | head -n 20

This will list the top 20 most frequent 15-bp sequences at the start of Read 2. Compare these to your CMO sequences (e.g., ATGAGGAATTCCTGC for CMO301). If none match, the library does not contain valid CMO tags—contact your sequencing facility to confirm the correct FASTQ assignment or redo the library preparation.

If the sequences appear but with low frequency, increase sequencing depth for the Multiplexing Capture library (aim for at least 5,000 reads per cell).

Your SLURM script and other configurations appear correct otherwise.

Thank you.

Kevin

• 0 views • link
updated by GenoMax GenoMax • written by Kevin Blighe Kevin Blighe

Thank you. I truly appreciate your remarkable attention to detail and your thoughtful support. I’ve got this;

multi*_R2_*.fastq.gz | awk 'NR%4 == 2 {print substr($1, 1, 15)}' | sort | uniq -c | sort -nr | head -n 20

5678151 AAGCAGTGGTATCAA
 688409 AGCAGTGGTATCAAC
 612361 GCAGTGGTATCAACG
 478532 GGGGGGGGGGGGGGG
 164270 CAGTGGTATCAACGC
  96590 GTGGTATCAACGCAG
  81280 AGTGGTATCAACGCA
  55840 GGTATCAACGCAGAG
  54976 AGGGGGGGGGGGGGG
  42937 TGGGGGGGGGGGGGG
  32736 ACTAAAAATACAAAA
  32501 AACGCAGAGTACATG
  31204 CTAAAAATACAAAAA
  25789 CAGCGTCAGATGTGT
  22723 AAAAAAAAAAAAAAA
  22524 GAGCAGTGGTATCAA
  22413 CCAGCACTTTGGGAG
  18291 TATCAACGCAGAGTA
  17739 ATCAACGCAGAGTAC
  17654 AAAAATACAAAAATT

None of these sequences match any of the known 10x CMO sequences (for example: ATGAGGAATTCCTGC, CATGCCAATAGAGCG, etc.).

I checked for gex and I got this

 zcat gex*.fastq.gz | awk 'NR%4 == 2 {print
substr($1, 1, 15)}' | sort | uniq -c | sort -nr | head -n 20
38282534 CGGATTCCACATCAT
31103566 CATGCCAATAGAGCG
30575807 ATGAGGAATTCCTGC
30118025 GTTGATCTATAACAG
29856109 AAGATGAGGTCTGTG
28247166 CCGTCGTCCAAGCAT
26181683 CGCGATATGGTCGGA
18364664 AACGTTAATCACTCA
16856734 AAGCTCGTTGGAAGA
 748009 GGGGGGGGGGGGGGG
 538319 CATGCCAATAGAGCA
 514467 AAGATGAGGTCTGTA
 410413 CGCGATATGGTCGGG
 395318 CGGATTCCACATCAA
 346860 ATGAGGAATTCCTGA
 324835 CGGATTCCACATCAG
 310981 CCGTCGTCCAAGCAA
 260718 CGGATTCCACATCAC
 257785 AAGCTCGTTGGAAGG
 250820 AACGTTAATCACTCG
• 0 views • link
written by zizigolu zizigolu

Thanks - I never forget a face [or, an avatar, in this case]...

Your examination of the FASTQ files indicates that the libraries specified in your multi_config.csv file are swapped. The sequences extracted from the multiplexing capture FASTQ files (HB216_multip_C4-SCI7T028-SCI5T028_233V53LT3) begin with adapter-like motifs, such as AAGCAGTGGTATCAA, which corresponds to the 10x reverse transcription primer sequence (AAGCAGTGGTATCAACGCAGAGT). This suggests that these files contain gene expression reads, where low-content or empty droplet reads often start with adapter sequences. Additionally, the presence of poly-G stretches (GGGGGGGGGGGGGGG) is consistent with over-sequencing artefacts in gene expression libraries.

In contrast, the sequences from the gene expression FASTQ files (HB216_gene_exp_C4-AK39983-AK40037_233V53LT3) match the expected 15-base pair CMO barcodes defined in your cmo_reference.csv file, such as CGGATTCCACATCAT (CMO308), CATGCCAATAGAGCG (CMO302), and others. This indicates that these files actually contain the multiplexing capture reads, where the first 15 bases of Read 2 correspond to the CMO tags.

To resolve the deplex error, revise your multi_config.csv file by swapping the feature_types in the [libraries] section as follows:

[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,Multiplexing Capture
HB216_multip_C4-SCI7T028-SCI5T028_233V53LT3,/users/scratch/fastq_files/X204SC25096963-Z01-F001_01/01.RawData/HB216_multip_C4,Gene Expression

Retain the cmo-set under [gene-expression], as specified in the Cell Ranger documentation. Rerun cellranger multi with the updated configuration. This adjustment should allow detection of the CMO tags in the correct multiplexing capture library.

Kevin

• 0 views • link
written by Kevin Blighe Kevin Blighe

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