https://cli.github.com/ (worth using if you aren't already)

nf-core pipelines download pinellolab/CRISPR_Pipeline --container-system singularity

that works on valid nf-core workflows to download the singularity images without running the workflow

example cluster config

singularity {

  enabled = true
  autoMounts = true
  cacheDir = "${launchDir}/singularity_images/"

}
executor {

  submitRateLimit = "2 sec"

}

process {

  executor = "slurm"
  scratch = true
  scratch = "${launchDir}/tmp"
}

How about this?

a test for fq/subsample:

PixelArt is fun

@chaochao wong, you certainly don't need a personal introduction to reach out to Fritz, but I'd be more than happy to do it. They are a great group. https://www.nature.com/articles/s41467-025-67237-y

SV hackathon https://www.linkedin.com/posts/fritz-sedlazeck-1b879370_sv-hackathon-2025-activity-7321240635405922305-8Xtx that is for 2025 -- id keep an eye on his slack for the 2026 hackathon in you're interested

* linkedin

I ran locally and got this error:

nextflow run pinellolab/CRISPR_Pipeline \

-r main \

-c ~/Downloads/config_hackathon_2026.js \

--outdir results_hackathon \

--input ~/Downloads/samplesheet.tsv

N E X T F L O W ~ version 25.10.4 Launching

<https://github.com/pinellolab/CRISPR_Pipeline>

[crazy_cajal] DSL2 - revision: dc93403d6e [main] WARN: Unrecognized config option ‘process.publishDir.path’ WARN: Unrecognized config option ‘process.publishDir.mode’ WARN: Unrecognized config option ‘process.publishDir.saveAs’ WARN: Access to undefined parameter

validate_params

-- Initialise it to a default value eg.

params.validate_params = some_value

[- ] NFC…R_PIPELINEseqSpecCheck pipelineguideWorkflow:seqSpecCheck - [- ] NFC…CRISPR PIPELINEprepare_mapping_pipeline:prepare_covariate - [- ] NFC…E_CRISPRCRISPR PIPELINEmapping_rna_pipeline:seqSpecParser - [- ] NFC…R_PIPELINEseqSpecCheck pipelineguideWorkflow:seqSpecCheck - [- ] NFC…CRISPR PIPELINEprepare_mapping_pipeline:prepare_covariate - [- ] NFC…E_CRISPRCRISPR PIPELINEmapping_rna_pipeline:seqSpecParser - [- ] NFC…ISPRCRISPR PIPELINEmapping_rna_pipeline:downloadReference | 0 of 1 [- ] NFCORE_CRISPRCRISPR PIPELINEmapping_rna_pipeline:mappingscRNA - [- ] NFC…_CRISPRCRISPR PIPELINEmapping_rna_pipeline:anndata_concat - [- ] NFC…CRISPRCRISPR PIPELINEmapping_guide_pipeline:seqSpecParser - [- ] NFC…RISPRCRISPR PIPELINEmapping_guide_pipeline:createGuideRef - [- ] NFC…_CRISPRCRISPR PIPELINEmapping_guide_pipeline:mappingGuide - [- ] NFC…RISPRCRISPR PIPELINEmapping_guide_pipeline:anndata_concat - [- ] NFC…PRCRISPR PIPELINEpreprocessing_pipeline:PreprocessAnnData - [- ] NFC…E_CRISPRCRISPR PIPELINEpreprocessing_pipeline:downloadGTF | 0 of 1 [- ] NFCORE_CRISPRCRISPR PIPELINECreateMuData - [- ] NFC…RISPR_PIPELINEguide assignment pipelineprepare_assignment - [- ] NFC…PIPELINEguide assignment pipelineguide_assignment_sceptre - [- ] NFC…SPRCRISPR PIPELINEguide_assignment_pipeline:mudata_concat - [- ] NFC…CRISPR PIPELINEinference_pipeline:prepare_guide_inference - [- ] NFC…PRCRISPR PIPELINEinference_pipeline:sceptre_chunk_prepare - [- ] NFC…CRISPRCRISPR PIPELINEinference_pipeline:inference_sceptre - [- ] NFC…ISPRCRISPR PIPELINEinference_pipeline:sceptre_chunk_merge - [- ] NFC…RISPRCRISPR PIPELINEinference_pipeline:inference_perturbo - [- ] NFCORE_CRISPRCRISPR PIPELINEinference_pipeline:mergedResults - [- ] NFC…CRISPR_PIPELINEinference pipelineinference_perturbo_trans - [- ] NFCORE_CRISPRCRISPR PIPELINEinference_pipeline:mergeMudata - Plus 4 more processes waiting for tasks… Sample: sample_null_null, Single-end: true, Files: [null, null] -[null] Pipeline completed with errors- ERROR ~ Cannot invoke method toLowerCase() on null object -- Check ‘.nextflow.log’ file for details ERROR ~ Pipeline failed. Please refer to troubleshooting docs: https://nf-co.re/docs/usage/troubleshooting -- Check ‘.nextflow.log’ file for details

@bioinfo lucas I created PR #74 to be merge to the nfcore_migration branch, which adds

subworkflows/local/fastq_subsample

that: 1. takes the pipeline input channel, 2. reformats it into the expected input channel for

fq_subsample

, and 3. runs the

fq_subsample

module from

modules/nf-core

. Here is an example

<http://subsample.nf|subsample.nf>

file to test this subworkflow:

#!/usr/bin/env nextflow


nextflow.enable.dsl = 2

project_dir = '/data/hps/assoc/private/sarthy_lab/user/cwo11/projects/CRISPR_Pipeline'
include { FASTQ_SUBSAMPLE } from '/data/hps/assoc/private/sarthy_lab/user/cwo11/projects/CRISPR_Pipeline/subworkflows/local/fastq_subsample'

// --- Parameters ---
params.input                   = "${project_dir}/tests/csv/test_samplesheet.csv"
params.subsample_seed          = 13
params.subsample_probability   = 0.1
params.subsample_record_count  = 1000
params.outdir   = "./subsample"

// --- Main Workflow ---
workflow {
    // 1. Create channel from CSV
    // this is exactly CRIRPR_PIPELINE prepares the ch_inputs (copy from subworkflow/local/utils_nfcore_crispr_pipeline)
    ch_inputs = channel.fromPath(params.input)
        .splitCsv(header: true)
        .map { row ->
            // FIXED: Added missing comma after modality
            def meta = [ // this is what defined in utils_nfc0re_crispr_pipeline
                id: "sample_${row.file_modality}_${row.measurement_sets}",
                single_end: false,
                modality: row.file_modality,
                measurement_sets: row.measurement_sets,
                sequencing_run: row.sequencing_run,
                lane: row.lane,
                seqspec: row.seqspec,
                barcode_onlist: row.barcode_onlist,
                guide_design: row.guide_design,
                barcode_hashtag_map: row.barcode_hashtag_map
            ]
            def r1_path = row.R1_path ? file(row.R1_path) : null
            def r2_path = row.R2_path ? file(row.R2_path) : null

            // Return structured tuple with meta and file paths
            [ meta, r1_path, r2_path ]
        }

    // 2. Run the subsampling subworkflow
    FASTQ_SUBSAMPLE(ch_inputs)

    // 3. DEBUGGING: print out simple output logging
    FASTQ_SUBSAMPLE.out.reads.view { meta, r1_path, r2_path ->
        "Finished subsampling ${meta.id} (${meta.modality}): ${[r1_path, r2_path].findAll { it }.join(', ')}"
    }

It can be run by

nextflow run <http://subsample.nf|subsample.nf> -c <your-path-to-CRISPR_PIPELINE>/conf/moduels/subsample.config

I run again with the local fastq files.

nextflow run pinellolab/CRISPR_Pipeline \

-r main \

-c config_hackathon_2026.js \

--outdir results_hackathon \

--input samplesheet.tsv

Nextflow 26.04.0 is available - Please consider updating your version to it N E X T F L O W ~ version 25.10.4 NOTE: Your local project version looks outdated - a different revision is available in the remote repository [6206bd632c] Launching

<https://github.com/pinellolab/CRISPR_Pipeline>

[soggy_yalow] DSL2 - revision: dc93403d6e [main] WARN: Unrecognized config option ‘process.publishDir.path’ WARN: Unrecognized config option ‘process.publishDir.mode’ WARN: Unrecognized config option ‘process.publishDir.saveAs’ WARN: Access to undefined parameter

validate_params

-- Initialise it to a default value eg.

params.validate_params = some_value

Sample: sample_null_null, Single-end: true, Files: [null, null] -[null] Pipeline completed with errors- ERROR ~ Cannot invoke method toLowerCase() on null object -- Check ‘.nextflow.log’ file for details ERROR ~ Pipeline failed. Please refer to troubleshooting docs: https://nf-co.re/docs/usage/troubleshooting -- Check ‘.nextflow.log’ file for details

NXF_VER=26.03.2-edge

I run on cluster:

NXF_VER=26.03.2-edge nextflow run <http://main.nf|main.nf> -profile test --outdir results/

Nextflow 26.03.4-edge is available - Please consider updating your version to it /share/pkg.8/nextflow/24.04.4/install/bin/nextflow: line 175: exec: : not found

1. go to workflow/crispr_pipeline/main.nf: add

include { FASTQ_SUBSAMPLE        } from '../../subworkflows/local/fastq_subsample'

NXF_VER=26.03.2-edge nextflow run <http://main.nf|main.nf> \

>     -profile test,singularity \

>     -c cluster.config \

>     --outdir results/ \

>     -resume

ERROR ~ Error executing process > ‘NFCORE_CRISPRCRISPR PIPELINEmapping_rna_pipeline:mappingscRNA (1)’ Caused by: Missing output file(s)

*_ks_transcripts_out/counts_unfiltered/adata.h5ad

expected by process

NFCORE_CRISPR:CRISPR_PIPELINE:mapping_rna_pipeline:mappingscRNA (1)

Command executed: echo “Processing A with IGVFFI1526NFSS.fastq.gz IGVFFI9278DMWO.fastq.gz” chemistry=$(extract_parsed_seqspec.py --file rna_parsed_seqSpec.txt) kb count -i transcriptome_index.idx -g transcriptome_t2g.txt --verbose -w barcode_file.txt \ --h5ad -x $chemistry -o A_ks_transcripts_out -t 8 \ IGVFFI1526NFSS.fastq.gz IGVFFI9278DMWO.fastq.gz --overwrite echo “scRNA KB mapping Complete” Command exit status: 0 Command output: Processing A with IGVFFI1526NFSS.fastq.gz IGVFFI9278DMWO.fastq.gz scRNA KB mapping Complete Command error: [progress] 59M reads processed (88.4% mapped) [progress] 60M reads processed (88.4% mapped) [progress] 61M reads processed (88.4% mapped) [progress] 62M reads processed (88.5% mapped) [progress] 63M reads processed (88.5% mapped) [progress] 65M reads processed (88.5% mapped) [progress] 66M reads processed (88.5% mapped) [progress] 67M reads processed (88.5% mapped) [progress] 68M reads processed (88.6% mapped) [progress] 69M reads processed (88.6% mapped) [progress] 70M reads processed (88.6% mapped) [progress] 71M reads processed (88.6% mapped) [progress] 72M reads processed (88.6% mapped) [progress] 73M reads processed (88.6% mapped) [progress] 74M reads processed (88.7% mapped) [progress] 75M reads processed (88.7% mapped) [progress] 76M reads processed (88.7% mapped) [progress] 77M reads processed (88.7% mapped) [progress] 78M reads processed (88.7% mapped) [progress] 79M reads processed (88.7% mapped) [progress] 80M reads processed (88.7% mapped) executor > local (10) [c2/10ac7d] NFCORE_CRISPRCRISPR PIPELINEseqSpecCheck_pipelineguideWorkflowseqSpecCheck [100%] 1 of 1 ✔️ [61/d03a9b] NFCORE_CRISPRCRISPR PIPELINEprepare_mapping_pipeline:prepare_covariate [100%] 1 of 1 ✔️ [cd/dfeddf] NFCORE_CRISPRCRISPR PIPELINEmapping_rna_pipeline:seqSpecParser [100%] 1 of 1 ✔️ [ca/211b3f] NFCORE_CRISPRCRISPR PIPELINEmapping_rna_pipeline:downloadReference [100%] 1 of 1 ✔️ [16/484e57] NFCORE_CRISPRCRISPR PIPELINEmapping_rna_pipeline:mappingscRNA (1) [ 0%] 0 of 1 ✘ [- ] NFCORE_CRISPRCRISPR PIPELINEmapping_rna_pipeline:anndata_concat - [57/d9d2c4] NFCORE_CRISPRCRISPR PIPELINEmapping_guide_pipeline:seqSpecParser [100%] 1 of 1 ✔️ [82/c95755] NFCORE_CRISPRCRISPR PIPELINEmapping_guide_pipeline:createGuideRef [100%] 1 of 1 ✔️ [e4/6d2b65] NFCORE_CRISPRCRISPR PIPELINEmapping_guide_pipeline:mappingGuide (A) [100%] 1 of 1 ✔️ [29/17ff03] NFCORE_CRISPRCRISPR PIPELINEmapping_guide_pipeline:anndata_concat [100%] 1 of 1 ✔️ [- ] NFCORE_CRISPRCRISPR PIPELINEpreprocessing_pipeline:PreprocessAnnData - [bd/b7d461] NFCORE_CRISPRCRISPR PIPELINEpreprocessing_pipeline:downloadGTF [100%] 1 of 1 ✔️ [- ] NFCORE_CRISPRCRISPR PIPELINECreateMuData - [- ] NFCORE_CRISPRCRISPR PIPELINEguide_assignment_pipeline:prepare_assignment - [- ] NFCORE_CRISPRCRISPR PIPELINEguide_assignment_pipeline:guide_assignment_sceptre - [- ] NFCORE_CRISPRCRISPR PIPELINEguide_assignment_pipeline:mudata_concat - [- ] NFCORE_CRISPRCRISPR PIPELINEinference_pipeline:prepare_guide_inference - [- ] NFCORE_CRISPRCRISPR PIPELINEinference_pipeline:sceptre_chunk_prepare - [- ] NFCORE_CRISPRCRISPR PIPELINEinference_pipeline:inference_sceptre - [- ] NFCORE_CRISPRCRISPR PIPELINEinference_pipeline:sceptre_chunk_merge - [- ] NFCORE_CRISPRCRISPR PIPELINEinference_pipeline:inference_perturbo - [- ] NFCORE_CRISPRCRISPR PIPELINEinference_pipeline:mergedResults - [- ] NFCORE_CRISPRCRISPR PIPELINEinference_pipeline:inference_perturbo_trans - [- ] NFCORE_CRISPRCRISPR PIPELINEinference_pipeline:mergeMudata - [- ] NFCORE_CRISPRCRISPR PIPELINEevaluation_pipeline:evaluation_plot - [- ] NFCORE_CRISPRCRISPR PIPELINEevaluation_pipeline:evaluation_controls - [- ] NFCORE_CRISPRCRISPR PIPELINEadditional_qc_plots - [- ] NFCORE_CRISPRCRISPR PIPELINEdashboard_pipeline:createDashboard_default - -[null] Pipeline completed with errors- ERROR ~ Error executing process > ‘NFCORE_CRISPRCRISPR PIPELINEmapping_rna_pipeline:mappingscRNA (1)’ Caused by: Missing output file(s)

*_ks_transcripts_out/counts_unfiltered/adata.h5ad

expected by process

NFCORE_CRISPR:CRISPR_PIPELINE:mapping_rna_pipeline:mappingscRNA (1)

Command executed: echo “Processing A with IGVFFI1526NFSS.fastq.gz IGVFFI9278DMWO.fastq.gz” chemistry=$(extract_parsed_seqspec.py --file rna_parsed_seqSpec.txt) kb count -i transcriptome_index.idx -g transcriptome_t2g.txt --verbose -w barcode_file.txt \ --h5ad -x $chemistry -o A_ks_transcripts_out -t 8 \ IGVFFI1526NFSS.fastq.gz IGVFFI9278DMWO.fastq.gz --overwrite echo “scRNA KB mapping Complete” Command exit status: 0 Command output: Processing A with IGVFFI1526NFSS.fastq.gz IGVFFI9278DMWO.fastq.gz scRNA KB mapping Complete Command error: [progress] 59M reads processed (88.4% mapped) [progress] 60M reads processed (88.4% mapped) [progress] 61M reads processed (88.4% mapped) [progress] 62M reads processed (88.5% mapped) [progress] 63M reads processed (88.5% mapped) [progress] 65M reads processed (88.5% mapped) [progress] 66M reads processed (88.5% mapped) [progress] 67M reads processed (88.5% mapped) [progress] 68M reads processed (88.6% mapped) [progress] 69M reads processed (88.6% mapped) [progress] 70M reads processed (88.6% mapped) [progress] 71M reads processed (88.6% mapped) [progress] 72M reads processed (88.6% mapped) [progress] 73M reads processed (88.6% mapped) [progress] 74M reads processed (88.7% mapped) [progress] 75M reads processed (88.7% mapped) [progress] 76M reads processed (88.7% mapped) [progress] 77M reads processed (88.7% mapped) [progress] 78M reads processed (88.7% mapped) [progress] 79M reads processed (88.7% mapped) [progress] 80M reads processed (88.7% mapped) [progress] 81M reads processed (88.8% mapped) [progress] 82M reads processed (88.8% mapped) [progress] 83M reads processed (88.8% mapped) [progress] 84M reads processed (88.8% mapped) [progress] 85M reads processed (88.8% mapped) [progress] 87M reads processed (88.8% mapped) [progress] 88M reads processed (88.8% mapped) [progress] 89M reads processed (88.8% mapped) [2026-04-29 163504,009] ERROR [main] An exception occurred Traceback (most recent call last): File “/opt/conda/envs/nfenv/lib/python3.11/site-packages/kb_python/main.py”, line 1933, in main COMMAND_TO_FUNCTION[args.command](parser, args, temp_dir=temp_dir) File “/opt/conda/envs/nfenv/lib/python3.11/site-packages/kb_python/main.py”, line 715, in parse_count count( File “/opt/conda/envs/nfenv/lib/python3.11/site-packages/ngs_tools/logging.py”, line 62, in inner return func(*args, **kwargs) ^^^^^^^^^^^^^^^^^^^^^ File “/opt/conda/envs/nfenv/lib/python3.11/site-packages/kb_python/count.py”, line 1350, in count bus_result = kallisto_bus( ^^^^^^^^^^^^^ File “/opt/conda/envs/nfenv/lib/python3.11/site-packages/kb_python/count.py”, line 244, in kallisto_bus run_executable(command) File “/opt/conda/envs/nfenv/lib/python3.11/site-packages/kb_python/dry/__init__.py”, line 25, in inner return func(*args, **kwargs) ^^^^^^^^^^^^^^^^^^^^^ File “/opt/conda/envs/nfenv/lib/python3.11/site-packages/kb_python/utils.py”, line 203, in run_executable raise sp.CalledProcessError(p.returncode, ' ‘.join(command)) subprocess.CalledProcessError: Command ‘/opt/conda/envs/nfenv/lib/python3.11/site-packages/kb_python/bins/linux/kallisto/kallisto bus -i transcriptome_index.idx -o A_ks_transcripts_out -x 0,0,160,16,261,0,92 -t 8 IGVFFI1526NFSS.fastq.gz IGVFFI9278DMWO.fastq.gz’ died with <Signals.SIGKILL: 9>. [2026-04-29 163504,020] DEBUG [main] Removing

A_ks_transcripts_out/tmp

directory Work dir: /Chris/CRISPR_Pipeline/work/16/484e57f0be298a98233a53f1bad09f Container: /Chris/CRISPR_Pipeline/work/singularity/ghcr.io-pinellolab-crispr_pipeline-conda-docker-latest.img Tip: you can replicate the issue by changing to the process work dir and entering the command

bash .command.run

-- Check ‘.nextflow.log’ file for details

https://compbiocore.github.io/VariantVisualization.jl/latest/#:~:text=VIVA%20is%20a%20user%2Dfriendly%20command,Variant%20Call%20Format%20(VCF)%20files.

@chaochao wong Trying to debug this problem

image.png

ok! it was asking for a partial execution and then collect the output

Hi all, I am Fabio (from clock.health), nice to join the team! I have been working with perturb-seq datasets in the past couple of years and would love to help on the project if there is any need