We received 10x single cell Flex data from a collaborator, it appears to have been run with

$ /cellranger/cellranger-7.1.0/cellranger multi \
    --id=${sample_id} \
    --csv=/${sample_id}.csv

$ cat ${sample_id}.csv
[gene-expression]
reference,/10xgenomics/refdata-gex-GRCh38-2020-A
probe-set,/cellranger/cellranger-7.1.0/probe_sets/Chromium_Human_Transcriptome_Probe_Set_v1.0.1_GRCh38-2020-A.csv

[libraries]
fastq_id,fastqs,feature_types
${sample_id},/FASTQS/outs/fastq_path/${sample_id}/,Gene Expression

I am trying to map these to nf-core/scrnaseq parameters

--aligner cellrangermulti \
--cellranger_vdj_index /10xgenomics/refdata-gex-GRCh38-2020-A \
--gex_frna_probe_set /cellranger/cellranger-7.1.0/probe_sets/Chromium_Human_Transcriptome_Probe_Set_v1.0.1_GRCh38-2020-A.csv \
...

I am not sure what VDJ means though. If

--cellranger_index

is also used for cellrangermulti, is that more appropriate? Is there anything else I am missing?

I have a question about the anndata → Seurat conversion. Currently, the conversion fails if the resulting sparse matrix exceeds ~2^31 non‑zero entries (R’s Matrix package limit). This usually only surfaces when combining the datasets at the end. I know that very large datasets can be handled via BPCells‑backed Seurat objects, but these require absolute paths and are harder to share in a core‑facility setting. Is there a better way to store this size as an Seurat object? (Currently, the process is just turned off, but would be nice to have the conversion included in the future)

Curious, I see

cellbender_removebackground

can run on GPU via

--cuda

. To set this in nf-core/scrnaseq, all I have to do is include

-profile gpu

when calling Nextflow?

Hello I have not run the scrnaseq pipeline yet, just looking through the docs to understand things. A quick question I have for the cellranger modules is do they produce the standard web summary reports at the end of their processing as it would normally?

Hi all. I’d like to ask about a possible thesis contribution to nf-core/scrnaseq around 10x CITE-seq via cellranger multi. Is #CHN5BV5DW the right place for that discussion? Where can I propose such an addition?

Any suggestions for ways to collaborate with @Kevin RUE ?

Hi all, there was this issue posted some time back about using single-end data: https://github.com/nf-core/scrnaseq/issues/394 I just got some single-end data, but the pipeline is unhappy even if I leave a blank spot for "fastq_2". Is there any way I can use this pipeline for single-end?

I have a question: What's the intended way to run the pipeline with 10x Flex data? I'm using the following nf-params, but it seems as if the probe set is not properly passed on to the multi config (frna_probeset = "".replace("./", work_dir) ), and it errors out. When I run it without cellranger_index, it also gives an error (although the index is not actually required as far as I can see it). What am I missing? 😄

{
  "input": "samplesheet.csv",
  "aligner": "cellrangermulti",
  "gex_frna_probe_set": "Chromium_Human_Transcriptome_Probe_Set_v2.0.0_GRCh38-2024-A.csv",
  "cellranger_index": "refdata-gex-GRCh38-2024-A",
  "skip_cellrangermulti_vdjref": true,
  "protocol": "auto",
  "skip_cellbender": true,
  "skip_fastqc": false,
  "save_align_intermeds": false
}

Good Afternoon! I am attempting to run the scrnaseq pipeline on an inDropsv3 dataset, but am having trouble finding the correct format for my sample sheet. I am inheriting this data from a few years back, and as such, am just interpreting what I can from the files given to me. There are 4 fastq files associated with each sample, which leads me to conclude the version chemistry. However, from what I learned online, the scrnaseq pipeline's kallisto functionality only allows for 2 fstq files input per sample, which obviously doesn't match up with the data I am seeing. Is there perhaps a dev branch of the pipeline or an older branch that is compatible with this data format? Thank you!

❌ scrnaseq/test_full failed 😿 Run Name romantic_edison Duration 7m 26s Status ❌ Failed Error Message

1:54PM INF [seqera-fusion] setting up AWS Batch spot instance monitoring provider=aws-batch
1:54PM INF [seqera-fusion] instance metadata ami-id=ami-0acc5dd6b31082def instance-id=i-092ff780cb05e8743 instance-type=c6id.12xlarge
1:54PM INF [seqera-fusion] starting incremental snapshot timer interval=300000
	STAR --genomeDir STARIndex --readFilesIn pbmc8k_S1_L007_R2_001.fastq.gz,pbmc8k_S1_L008_R2_001.fastq.gz pbmc8k_S1_L007_R1_001.fastq.gz,pbmc8k_S1_L008_R1_001.fastq.gz --runThreadN 12 --outFileNamePrefix pbmc8k. --soloCBwhitelist whitelist.uncompressed.txt --soloType CB_UMI_Simple --soloFeatures Gene --soloUMIlen 10 --sjdbGTFfile genome_genes.gtf --outSAMattrRGline ID:pbmc8k SM:pbmc8k --soloCellFilter CellRanger2.2 10000 0.99 10 --readFilesCommand zcat --runDirPerm All_RWX --outWigType bedGraph --twopassMode Basic --outSAMtype BAM SortedByCoordinate --limitBAMsortRAM 77309411328
	STAR version: 2.7.10b   compiled: 2022-11-01T09:53:26-04:00 :/home/dobin/data/STAR/STARcode/STAR.master/source
May 28 13:54:13 ..... started STAR run
May 28 13:54:14 ..... loading genome
EXITING because of FATAL ERROR: Genome version: 20201 is INCOMPATIBLE with running STAR version: 2.7.10b
SOLUTION: please re-generate genome from scratch with running version of STAR, or with version: 2.7.4a
May 28 13:54:14 ...... FATAL ERROR, exiting
zcat: write error: Broken pipe
pbmc8k._STARtmp//readsCommand_read2: line 4: echo: write error: Broken pipe
zcat: write: Broken pipe
zcat: write error: Broken pipe
zcat: write: Broken pipe
zcat: write error: Broken pipe
zcat: write: Broken pipe
pbmc8k._STARtmp//readsCommand_read1: line 4: echo: write error: Broken pipe
zcat: write error: Broken pipe
zcat: write: Broken pipe
FUSION_GPU_USED=false
Fusion Info:
    available_disk: start:2.8TB, min:2.8TB, max:2.8TB, end:2.8TB
    clone_namespace: false
    download: 2293.56MB, drops:0, skip:0, sync:0, req:19
    fusion_version: 2.5.12-8ea351d
    kernel_version: 6.1
    max_open_files: 1048576
    upload: 11.96MB, dro...

Command Line

nextflow run '<https://github.com/nf-core/scrnaseq>' -name romantic_edison -params-file '<https://api.cloud.seqera.io/ephemeral/ar8SLDVM-kSYQinORabCFA.json>' -with-tower -r b7366482866bb64cf25a8c46940af2ab363698af -profile test_full

May 28, 2026 at 1:54 PM

❌ scrnaseq/test_full failed 😿 Run Name condescending_descartes Duration 9m 36s Status ❌ Failed Error Message

1:55PM INF [seqera-fusion] setting up AWS Batch spot instance monitoring provider=aws-batch
1:55PM INF [seqera-fusion] instance metadata ami-id=ami-0acc5dd6b31082def instance-id=i-0a717b0eba0163bf9 instance-type=m6id.12xlarge
1:55PM INF [seqera-fusion] starting incremental snapshot timer interval=300000
Thank you for using cellranger. To help us improve our product,
anonymized telemetry data has been collected and sent to 10X Genomics.
This data helps us understand usage patterns, diagnose issues,
and prioritize improvements.
You can inspect the telemetry metrics sent by looking in
/root/.cache/tenx/telemetry/cellranger/10.0.0
For more details on what data is collected and how it's used, please visit
<https://10xgen.com/pipeline-telemetry>
You can disable telemetry at any time by running the following command:
	cellranger telemetry disable
Martian Runtime - v4.0.14
Serving UI at <http://ip-172-31-5-11.eu-west-1.compute.internal:36541?auth=ywQsI3R6x0m7opAMYOj6ntkg5dZst0BbcepIkXa0HdQ>
Running preflight checks (please wait)...
2026-05-28 13:55:19 [runtime] (ready)           ID.mkref_cellranger_reference.MAKE_REFERENCE._MAKE_REFERENCE
2026-05-28 13:55:19 [runtime] (run:local)       ID.mkref_cellranger_reference.MAKE_REFERENCE._MAKE_REFERENCE.fork0.split
2026-05-28 13:55:19 [runtime] (split_complete)  ID.mkref_cellranger_reference.MAKE_REFERENCE._MAKE_REFERENCE
2026-05-28 13:55:19 [runtime] (run:local)       ID.mkref_cellranger_reference.MAKE_REFERENCE._MAKE_REFERENCE.fork0.join
2026-05-28 13:56:03 [runtime] (failed)          ID.mkref_cellranger_reference.MAKE_REFERENCE._MAKE_REFERENCE
[error] mkref has failed: error building reference package
Error detected in GTF file: Parsing GTF on line 284226: source: cannot contain spaces
Line = 'chr11	Curated Genomic	exon	5225466	5225726	.	-	.	gene_id "HBB"; gene_name "HBB"; p_id "P21712"; transcript_id "NM_000518.4"; tss_id "TSS44384";'
2026-05-28 13:56:03 Shutting down.
Saving pipestance info to "mkref_cellranger_reference/mkref_cel...

Command Line

nextflow run '<https://github.com/nf-core/scrnaseq>' -name condescending_descartes -params-file '<https://api.cloud.seqera.io/ephemeral/QevG8VwnlsSbhDs8zs4veQ.json>' -with-tower -r b7366482866bb64cf25a8c46940af2ab363698af -profile test_full

May 28, 2026 at 1:56 PM

❌ scrnaseq/test_full failed 😿 Run Name condescending_volhard Duration 2h 30m 47s Status ❌ Failed Error Message

3:50PM INF [seqera-fusion] setting up AWS Batch spot instance monitoring provider=aws-batch
3:50PM INF [seqera-fusion] instance metadata ami-id=ami-0acc5dd6b31082def instance-id=i-0919fd14efdf5d915 instance-type=c6id.8xlarge
3:50PM INF [seqera-fusion] starting incremental snapshot timer interval=300000
3:55PM INF [seqera-fusion] sending incremental snapshot signal (SIGUSR1)
4:00PM INF [seqera-fusion] sending incremental snapshot signal (SIGUSR1)
4:05PM INF [seqera-fusion] sending incremental snapshot signal (SIGUSR1)
4:10PM INF [seqera-fusion] sending incremental snapshot signal (SIGUSR1)
4:15PM INF [seqera-fusion] sending incremental snapshot signal (SIGUSR1)
FUSION_GPU_USED=false
4:17PM ERR [seqera-fusion] cannot upload part copy error="operation error S3: UploadPartCopy, https response error StatusCode: 400, RequestID: KPGJTTSEHY8K3637, HostID: An+Jt4m25zAMm478x81VLZ4zR7vvtxvGvpHKks2f1UPLndghyFTT8FePYQhZPHwu/O7JlKRuLhY=, api error InvalidRequest: The specified copy source is larger than the maximum allowable size for a copy source: 5368709120" goid=584266 id=/s3/nf-core-awsmegatests/work/scrnaseq/work-b7366482866bb64cf25a8c46940af2ab363698af/dd/7e122ffcef07ef9b7689fe3841e42d/simpleaf_quant/af_map/map.rad length=11274289152 offset=134217728
4:17PM ERR [seqera-fusion] cannot upload part copy error="operation error S3: UploadPartCopy, https response error StatusCode: 0, RequestID: , HostID: , canceled, context canceled" goid=584292 id=/s3/nf-core-awsmegatests/work/scrnaseq/work-b7366482866bb64cf25a8c46940af2ab363698af/dd/7e122ffcef07ef9b7689fe3841e42d/simpleaf_quant/af_map/map.rad.d8c6cscku2lc76n5mopg.12616466432.d8c68mcku2lc76n5mhag.chk length=134217728 offset=0
4:17PM ERR [seqera-fusion] cannot upload part copy error="operation error S3: UploadPartCopy, https response error StatusCode: 0, RequestID: , HostID: , canceled, context canceled" goid=584297 id=/s3/nf-core-awsmegatests/work/scrnaseq/work-b7366482866bb64cf25a8c46940af2ab363698af/dd/7e122ffcef07ef9b7689fe38...

Command Line

nextflow run '<https://github.com/nf-core/scrnaseq>' -name condescending_volhard -params-file '<https://api.cloud.seqera.io/ephemeral/vjgGGywhoYT1WoCtWKnXOA.json>' -with-tower -r b7366482866bb64cf25a8c46940af2ab363698af -profile test_full

May 28, 2026 at 4:17 PM

✅ scrnaseq/test_full completed successfully! 🎉 Run Name fabulous_hawking Duration 3h 11m 5s Status ✅ Success Tasks Completed: 18 May 28, 2026 at 4:57 PM

Hi everyone! I'm trying to run a pipeline but getting error that says there's no config file called test_multiome.config. And it seems it's indeed missing on git page. Is that a bug or am I doing something wrong?

Nvm, seems last version of nextflow is incompatible with pipeline. I reverted and it works fine

Hi all, I recently joined and I'm hoping to start contributing to nf-core/scrnaseq. I'm an evolutionary bio PhD student at UIUC who codes in Nextflow (also fluent in Python and Rust) day-to-day, currently getting set up (running the test profile, reading the contributing guide). Could anyone point me to a good-first-issue or a docs gap that'd make a sensible first contribution? Happy to claim something and follow the conventions. I just want to make sure I pick something up the right way.

Hey everyone - version 4.2.0 of #CHN5BV5DW was released over the weekend! It comes with: • Upgrade to cellranger v10 • Add support for strict syntax, which is required to make it work with nextflow 26.04 and beyond • Replace alevinqc with qcatch (simpleaf alignment route) • Improvements concerning reference genome handling • A couple of additional bugfixes More details: Full changelog

By any chance, is there still a way to pass boolean values successfully with the CLI? e.g. with

--skip_cellbender

?

Also this is quite minor, but for (at least) version 4.2.0, the links to the usage docs within the help bubbles of nf-co.re/scrnaseq/4.2.0/parameters link to nf-core/rnaseq rather than scrnaseq. Had me very confused for a moment. e.g.

--input

Final thing I hope: based on this commit it appears you should be able to specify whether or not to include intronic reads in cellranger count / multi using the sample sheet. However in cellranger multi this appears to be ignored:

WARN: Found the following unidentified headers in 260708_mini_nfcore_sample_sheet.csv:
        - include_introns

And the cellranger QC confirms this:

Include Introns true

(I set to false for all samples in the sample sheet) Is this expected behavior / user error, or should I open an issue?

I am getting this error on the latest release of the pipeline (I also posted this to Github, issue 567): Command exit status: 1 Command output: ERROR: failed to parse CSV at line: 1, col: 1 This is my command: nextflow run ./scrnaseq/main.nf -profile singularity,gpu \ --input samplesheet.csv \ --protocol auto \ --genome GRCh38 \ --aligner cellrangermulti \ --outdir outdir \ --gtf gencode.v49.annotation.gtf \ --cellranger_multi_barcodes multi_barcodes.csv \ --gex_target_panel Chromium_Human_Transcriptome_Probe_Set_v2.0.0_GRCh38-2024-A.csv \ --gex_reference_version GRCh38 \ --skip_cellrangermulti_vdjref \ -resume I suspect there is an issue with script in creating the proper config file for cellrangermulti: scrnaseq/modules/nf-core/cellranger/multi/templates/cellranger_multi.py

Computational Biologist | Cambridge, UK (Onsite Role) | Biotech I'm partnering with an exciting biotech in Cambridge and they are looking for a talented Computational Biologist to join their growing team. You'll be working across single-cell and methylation datasets, building production-grade pipelines and collaborating closely with wet-lab scientists to drive target discovery. You'll need: • scRNA-seq analysis experience (Scanpy, STARsolo) • Python proficiency + Linux/Bash/Git • Workflow language experience (Nextflow or Snakemake) • Strong biological understanding My email is christina.giakou@cavenaghhealth.com if you want to know more!

Hi! I am trying to use scrnaseq pipeline for GEX+TCR analysis with cellranger multi pipeline. I am struggling with the VDJ reference parameters. This is my launcher command:

NXF_VER=25.10.4 nextflow run nf-core/scrnaseq \
    --input samplesheet.csv \
    --outdir ../PROCESSING \
    -profile apptainer \
    --aligner cellrangermulti \
    --igenomes_base ~/Shared-MarcelCosta/references/cellranger-10.0.0 \
    --fasta ~/Shared-MarcelCosta/references/cellranger-10.0.0/genome.fa \
    --gtf ~/Shared-MarcelCosta/references/cellranger-10.0.0/genes.gtf \
    --cellranger_vdj_index ~/Shared-MarcelCosta/references/cellranger-10.0.0/regions.fa

And the pipeline tries to find the reference in the work directory in:

references/vdj/regions.fa/fasta/regions.fa

However, when I go to the work directory the reference is in:

references/vdj/regions.fa

Any clue if I should specify the reference in a different way? Thanks!!

Hi! Is there a way to introduce options into the cellranger multi config csv that is automatically generated by the pipeline? I have to disable the library compatibility check and error points me to do it there.

Anyone interested in version 10.1.0 update for cellranger? 10xgenomics.com/support/software/cell-ranger/downloads

It appears the docker container process is manual, I don't believe I have write access to the Dockerhub nfcore organization github.com/nf-core/modules/…/cellranger#…

Hi everyone, together with @Delfina Terradas we are going to be doing some maintainance to this pipeline (template updates, bugfixes, nf-core module updates). Please let us know if any current pipeline maintainers (other than Gregor or Felipe) want to be involved and tagged for review 🙂 Thank you!

I have a pull request in progress for cellranger 10.1.0 update, one of the

cellranger multi

tests is failing both locally and in Github Actions with

Test Process CELLRANGER_MULTI

  Test [af57fe63] 'cellranger - multi - 10k - PBMC' Assertion failed:
...
     >   2026-10-01 15:01:08 [runtime] (failed)          ID.subsampled_sc5p_v2_hs_PBMC_10k.SC_MULTI_CS.SC_MULTI_CORE.MULTI_GEM_WELL_PROCESSOR.COUNT_GEM_WELL_PROCESSOR._BASIC_SC_RNA_COUNTER._MATRIX_COMPUTER.MAKE_SHARD
      >
      >   [error] Pipestance failed. Error log at:
      >   subsampled_sc5p_v2_hs_PBMC_10k/SC_MULTI_CS/SC_MULTI_CORE/MULTI_GEM_WELL_PROCESSOR/COUNT_GEM_WELL_PROCESSOR/_BASIC_SC_RNA_COUNTER/_MATRIX_COMPUTER/MAKE_SHARD/fork0/chnk0-u00a9be75b4/_errors
      >
      >   Log message:
      >   There were no reads to process. Please check whether the read lengths in the input fastqs satisfy the minumum read length requirements for the chemistry.

github.com/nf-core/modules/…/110431458175?pr=13012

depictio Depictio dashboard for scrnaseq: your feedback wanted Hi all! 👋 I am building Depictio, an open-source platform from EMBL to explore pipeline results without writing a Shiny or Streamlit app each time. A pipeline gets a dashboard template: a short definition that turns the outputs of any run into an interactive dashboard. I am now sharing it more widely, so I would like the view of scrnaseq users and developers. ✨ What it does • MultiQC reports (aggregated across runs) next to result tables, images, phylogenetic trees, maps... with linked filters • Reusable: point the template at any new run of the same pipeline and you get the same dashboard • Fully editable: modify any dashboard from the GUI or in YAML, so the template is a starting point, not a constraint • Versioned, with comments and annotations on any component • Runs as a Python package, with Docker Compose or on Kubernetes, and can serve as a companion web app for a publication 🔬 For scrnaseq: library QC against 10x guidance, cell calling, agreement between Cell Ranger, simpleaf and kallisto, per-cell QC, UMAP and t-SNE, clusters, marker genes and a lasso comparison of two groups of cells, next to the MultiQC report. 👀 What I need: I have built 25 templates and want people who really use these pipelines to review and criticise them. What is useful? What is misleading? 👉 Dashboard: scrnaseq dashboard (no account needed) 💬 Comment on the dashboard (Google sign-in) or in the GitHub discussion dedicated to scrnaseq. ⭐ A star on the repo is welcome if you find it useful. Thanks! 🙏 🎤 In you're interested in the project, I was speaking about Depictio at the Nextflow Summit (session page), and the recording will be available in the coming weeks.

Hi all! New here, so apologies if this has come up before. My team wants to use nf-core/scrnaseq (v4.2.0) with cellranger multi to process multiplexed single-nucleus data from the 10x GEM-X Flex v2 protocol. I don’t see Flex v2 listed under

--protocol

in the usage docs. Since Flex v2 needs Cell Ranger ≥10.0, does anyone know whether 4.2.0 (or the dev branch) supports it? Thanks!