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fix probe-set inclusion assertion #12601

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fix probe-set inclusion assertion #12601

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c58e945
fix probe-set inclusion assertion
fmalmeida Aug 6, 2026
0843e28
add probe-set when mutually exclusive features are not available.
fmalmeida Aug 6, 2026
ad88241
remove trailling whitespace
fmalmeida Aug 6, 2026
f11c654
add test for "pipeline scenario"
fmalmeida Aug 6, 2026
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4 changes: 3 additions & 1 deletion modules/nf-core/cellranger/multi/main.nf
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Original file line number Diff line number Diff line change
Expand Up @@ -64,7 +64,9 @@ process CELLRANGER_MULTI {
if (has_gex) {
gex_section << '[gene-expression]'
gex_section << "reference,\$PWD/${gex_reference.name}"
if (gex_frna_probeset) gex_section << "probe-set,\$PWD/${gex_frna_probeset.name}"

// still allow frna probe-set for flex, but avoid adding when CMO or OCM barcodes are present, since those are mutually exclusive with frna
if (gex_frna_probeset && !has_cmo && !has_ocm) gex_section << "probe-set,\$PWD/${gex_frna_probeset.name}"

// GEX options forwarded from the gex_options input map
['filter-probes', 'r1-length', 'r2-length', 'chemistry', 'expect-cells', 'force-cells',
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116 changes: 116 additions & 0 deletions modules/nf-core/cellranger/multi/tests/main.nf.test
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Expand Up @@ -731,4 +731,120 @@ nextflow_process {
}

}
test("cellranger - multi - 10k - PBMC - with cmo - pipeline scenario where different references might be available for different samples") {

when {
process {
"""
//
// preparation: unfortunately have to repeat data load
//

/**********************************/
/*** stage 10k PBMC w/ CMO data ***/
/**********************************/

// stage 3' CMO FASTQ test data
cmo_fastqs_10k_pbmc_cmo = [
file(params.modules_testdata_base_path + 'genomics/homo_sapiens/10xgenomics/cellranger/10k_pbmc_cmo/fastqs/cmo/subsampled_SC3_v3_NextGem_DI_CellPlex_Human_PBMC_10K_1_multiplexing_capture_S1_L001_R1_001.fastq.gz', checkIfExists: true),
file(params.modules_testdata_base_path + 'genomics/homo_sapiens/10xgenomics/cellranger/10k_pbmc_cmo/fastqs/cmo/subsampled_SC3_v3_NextGem_DI_CellPlex_Human_PBMC_10K_1_multiplexing_capture_S1_L001_R2_001.fastq.gz', checkIfExists: true)
]
def cmo_fastq_samplename_10k_pbmc_cmo = "subsampled_SC3_v3_NextGem_DI_CellPlex_Human_PBMC_10K"

// stage 3' gene expression FASTQ test data
threepgex_fastqs_10k_pbmc_cmo = [
file(params.modules_testdata_base_path + 'genomics/homo_sapiens/10xgenomics/cellranger/10k_pbmc_cmo/fastqs/gex_1/subsampled_SC3_v3_NextGem_DI_CellPlex_Human_PBMC_10K_1_gex_S2_L001_R1_001.fastq.gz', checkIfExists: true),
file(params.modules_testdata_base_path + 'genomics/homo_sapiens/10xgenomics/cellranger/10k_pbmc_cmo/fastqs/gex_1/subsampled_SC3_v3_NextGem_DI_CellPlex_Human_PBMC_10K_1_gex_S2_L001_R2_001.fastq.gz', checkIfExists: true)
]
def threepgex_fastq_samplename_10k_pbmc_cmo = "subsampled_SC3_v3_NextGem_DI_CellPlex_Human_PBMC_10K"

// stage feature barcode reference for antibody capture
cmo_reference_10k_pbmc_cmo = file(params.modules_testdata_base_path + 'genomics/homo_sapiens/10xgenomics/cellranger/10k_pbmc_cmo/10k_pbmc_cmo_count_feature_reference.csv', checkIfExists: true)

// CMO needs a barcode file
cmo_barcodes_csv = file("cmo_barcodes.csv")
cmo_barcodes_csv.text = "sample_id,cmo_ids,description\\nPBMCs_human_1,CMO301,PBMCs_human_1\\nPBMCs_human_2,CMO302,PBMCs_human_2"


/**************************************/
/*** end stage 10k PBMC w/ CMO data ***/
/**************************************/

probeset = file(params.modules_testdata_base_path + 'genomics/homo_sapiens/10xgenomics/cellranger/references/flex/Chromium_Human_Transcriptome_Probe_Set_v1.1.0_GRCh38-2024-A.chr22.csv', checkIfExists: true)

// create empty channels to fill unused cellranger multi arguments
// fastqs need a [ meta, ref ] structure
// references just need a path
ch_gex_fastqs = [ [:], [], [:] ]
ch_vdj_fastqs = [ [:], [], [:] ]
ch_ab_fastqs = [ [:], [], [:] ]
ch_beam_fastqs = [ [:], [], [:] ]
ch_cmo_fastqs = [ [:], [], [:] ]
ch_crispr_fastqs = [ [:], [], [:] ]
ch_gex_frna_probeset = []
ch_gex_targetpanel = []
ch_vdj_reference = []
ch_vdj_primer_index = []
ch_fb_reference = []
ch_beam_antigen_panel = []
ch_beam_control_panel = []
ch_cmo_reference = []
ch_cmo_barcodes = []
ch_ocm_barcodes = []
ch_cmo_sample_assignment = []
ch_frna_sampleinfo = []

// collect references and fastq files for staging
ch_gex_fastqs_10k_pbmc_cmo = Channel.of( threepgex_fastqs_10k_pbmc_cmo )
.collect()
.map { reads -> [ [ id:threepgex_fastq_samplename_10k_pbmc_cmo ], reads, [ "expect-cells":"1000", chemistry:"SC3Pv3", "create-bam":false, "no-secondary":true ] ] }
ch_cmo_fastqs_10k_pbmc_cmo = Channel.of( cmo_fastqs_10k_pbmc_cmo )
.collect()
.map { reads -> [ [ id:cmo_fastq_samplename_10k_pbmc_cmo ], reads, [:] ] }
ch_cmo_reference_10k_pbmc_cmo = Channel.of( cmo_reference_10k_pbmc_cmo )

// CMO analysis needs barcodes
ch_cmo_barcodes_10k_pbmc_cmo = Channel.fromPath( cmo_barcodes_csv )

//
// execution
//
input[0] = [ id:'subsampled_SC3_v3_NextGem_DI_CellPlex_Human_PBMC_10K', single_end:false ]
input[1] = ch_gex_fastqs_10k_pbmc_cmo
input[2] = ch_vdj_fastqs
input[3] = ch_ab_fastqs
input[4] = ch_beam_fastqs
input[5] = ch_cmo_fastqs_10k_pbmc_cmo
input[6] = ch_crispr_fastqs
input[7] = CELLRANGER_MKREF.out.reference
input[8] = probeset
input[9] = ch_gex_targetpanel
input[10] = ch_vdj_reference
input[11] = ch_vdj_primer_index
input[12] = ch_fb_reference
input[13] = ch_beam_antigen_panel
input[14] = ch_beam_control_panel
input[15] = ch_cmo_reference_10k_pbmc_cmo
input[16] = ch_cmo_barcodes_10k_pbmc_cmo
input[17] = ch_cmo_sample_assignment
input[18] = ch_frna_sampleinfo
input[19] = ch_ocm_barcodes
input[20] = false // default to false to guarantee renaming during test
"""
}
}

then {
assert process.success
assert snapshot(
process.out.findAll { key, val -> key.startsWith("versions") },
process.out.outs[0][1].findAll { file(it).name == 'assignment_confidence_table.csv' },
process.out.outs[0][1].findAll { file(it).name == 'tag_calls_summary.csv' },
process.out.outs[0][1].findAll { file(it).name == 'cells_per_tag.json' },
process.out.outs[0][1].findAll { file(it).name.contains('metrics_summary.csv') },
process.out.outs[0][1].findAll { file(it).name.contains('sample_filtered_feature_bc_matrix.h5') }
).match()
}

}
}
35 changes: 35 additions & 0 deletions modules/nf-core/cellranger/multi/tests/main.nf.test.snap
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Original file line number Diff line number Diff line change
Expand Up @@ -37,6 +37,41 @@
"nextflow": "26.04.3"
}
},
"cellranger - multi - 10k - PBMC - with cmo - pipeline scenario where different references might be available for different samples": {
"content": [
{
"versions_cellranger": [
[
"CELLRANGER_MULTI",
"cellranger",
"10.0.0"
]
]
},
[
"assignment_confidence_table.csv:md5,1bccc6e7e9105f12f938d61ec6f94d3c"
],
[
"tag_calls_summary.csv:md5,55d2c04c1426c3f7a1d9282a9948ba6a"
],
[
"cells_per_tag.json:md5,3f28cb95f291228519bb80734c7ed570"
],
[
"metrics_summary.csv:md5,59a1506efa32223938f9cb097cdc4451",
"metrics_summary.csv:md5,d0d4d616e02ccfd67566ffe44decf5d6"
],
[
"sample_filtered_feature_bc_matrix.h5:md5,fbb622a39ed07c95ce880f2b7b0f96e8",
"sample_filtered_feature_bc_matrix.h5:md5,9da06141abdf8262d5115fc88e5eb3f2"
]
],
"timestamp": "2026-08-06T09:44:42.817483788",
"meta": {
"nf-test": "0.9.5",
"nextflow": "26.04.6"
}
},
"cellranger - multi - 10k - PBMC - with cmo": {
"content": [
{
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