diff --git a/CHANGELOG.md b/CHANGELOG.md index d181a907..4ff383ef 100644 --- a/CHANGELOG.md +++ b/CHANGELOG.md @@ -3,6 +3,12 @@ The format is based on [Keep a Changelog](https://keepachangelog.com/en/1.0.0/) and this project adheres to [Semantic Versioning](https://semver.org/spec/v2.0.0.html). +## [Unreleased] + +### Features + +- Fix [#375](https://github.com/nf-core/scrnaseq/issues/375): mismatch between index and probeset when cellranger multi is used without a prebuilt index and an FFPE probeset is passed ([#502](https://github.com/nf-core/scrnaseq/pull/502)) + ## v4.1.0 - 2025-08-01 ### Features diff --git a/assets/frna_probeset_subset.csv b/assets/frna_probeset_subset.csv index 84772fa0..35800de8 100644 --- a/assets/frna_probeset_subset.csv +++ b/assets/frna_probeset_subset.csv @@ -1,7 +1,7 @@ #probe_set_file_format=2.0 #panel_name=Chromium Human Transcriptome Probe Set v1.0.1 #panel_type=predesigned -#reference_genome=gex_reference +#reference_genome=GRCh38 #reference_version=2020-A gene_id,probe_seq,probe_id,included,region ENSG00000000003,GGTGACACCACAACAATGCAACGTATTTTGGATCTTGTCTACTGCATGGC,ENSG00000000003|TSPAN6|8eab823,TRUE,spliced diff --git a/conf/test_cellranger_multi.config b/conf/test_cellranger_multi.config index 7684a49b..d48df652 100644 --- a/conf/test_cellranger_multi.config +++ b/conf/test_cellranger_multi.config @@ -46,6 +46,7 @@ profiles { cellranger_multi_barcodes = "${projectDir}/assets/cellranger_barcodes_samplesheet.csv" gex_frna_probe_set = "${projectDir}/assets/frna_probeset_subset.csv" fb_reference = "${projectDir}/assets/fb_reference.csv" + gex_reference_version = "GRCh38" // Genome references fasta = 'https://ftp.ensembl.org/pub/release-110/fasta/homo_sapiens/dna/Homo_sapiens.GRCh38.dna.chromosome.14.fa.gz' diff --git a/docs/usage.md b/docs/usage.md index e77d47bf..bd9b78e3 100644 --- a/docs/usage.md +++ b/docs/usage.md @@ -282,8 +282,10 @@ If you are using cellranger-multi you have to add the column _feature_type_ to i > When running cellranger multi, without any VDJ data, users can also skip VDJ automated ref building with: `--skip_cellrangermulti_vdjref`. -- When working with **FFPE data**, a prob set needs to be specified via `--gex_frna_probe_set`. This file is typically - [provided by 10x](https://www.10xgenomics.com/support/software/cell-ranger/downloads#probe-set-downloads). E.g. [testing ffpe probe set](../assets/frna_probeset_subset.csv). +- When working with **FFPE data**: + - a probe set needs to be specified via `--gex_frna_probe_set`. This file is typically + [provided by 10x](https://www.10xgenomics.com/support/software/cell-ranger/downloads#probe-set-downloads). E.g. [testing ffpe probe set](../assets/frna_probeset_subset.csv). + - a GEX reference genome version (e.g. GRCh38, GRCm39) via `--gex_reference_version` must be specified unless a pre-built index is provided via `--cellranger_index`. This **must** match the reference in the probe set, which can be found in the header. - When working with **Cell Multiplexing Oligos (CMOs)**, a reference file can to be provided via `--gex_cmo_set`. The default reference file, as well as a description how to write a custom one, are [available from the 10x documentation](https://www.10xgenomics.com/support/software/cell-ranger/latest/analysis/running-pipelines/cr-3p-multi#cmo-ref). By default, the Cell Ranger's default CMO-set. diff --git a/nextflow.config b/nextflow.config index a5c20ca7..dadcd06f 100644 --- a/nextflow.config +++ b/nextflow.config @@ -59,6 +59,7 @@ params { vdj_inner_enrichment_primers = null gex_barcode_sample_assignment = null cellranger_multi_barcodes = null + gex_reference_version = null // Template Boilerplate options diff --git a/nextflow_schema.json b/nextflow_schema.json index 80e18e3f..e48b8f9c 100644 --- a/nextflow_schema.json +++ b/nextflow_schema.json @@ -330,6 +330,10 @@ "exists": true, "description": "Provide a probe set for fixed RNA-seq profiling (used with FFPE samples). Please refer to the [10x documentation about probesets](https://www.10xgenomics.com/support/single-cell-gene-expression-flex/documentation/steps/probe-sets/chromium-frp-probe-set-files) for more details." }, + "gex_reference_version": { + "type": "string", + "description": "Reference version (e.g. GRCh38, GRCm39). Required if `gex_frna_probe_set` is used and `cellranger_index` is not set. Must match the genome version in the `gex_frna_probe_set` file." + }, "gex_target_panel": { "type": "string", "description": "Provide a panel description for targeted sequencing.", diff --git a/subworkflows/local/align_cellrangermulti.nf b/subworkflows/local/align_cellrangermulti.nf index 3b12b02b..cc248ada 100644 --- a/subworkflows/local/align_cellrangermulti.nf +++ b/subworkflows/local/align_cellrangermulti.nf @@ -136,11 +136,38 @@ workflow CELLRANGER_MULTI_ALIGN { // if ( !cellranger_gex_index ) { + // Validate that gex_reference_version is provided when required + if ( params.gex_frna_probe_set && !params.gex_reference_version ) { + error "Parameter 'gex_reference_version' is required when 'gex_frna_probe_set' is provided and 'cellranger_index' is not provided. The reference genome version must match the probeset reference." + } + + // Validate that gex_reference_version matches the probeset reference genome + if ( params.gex_frna_probe_set && params.gex_reference_version ) { + def probeset_file = file(params.gex_frna_probe_set) + def probeset_reference = null + probeset_file.withReader { reader -> + String line + while ((line = reader.readLine()) != null) { + if (line.startsWith("#reference_genome=")) { + ref_split = line.split("=") + if (ref_split.size() > 1) { + probeset_reference = ref_split[1].trim() + } + break + } + } + } + if ( probeset_reference != params.gex_reference_version ) { + error "Parameter 'gex_reference_version' (${params.gex_reference_version}) does not match the probeset reference genome (${probeset_reference}). Please ensure the reference genome version matches the probeset file." + } + } + // Make reference genome + def reference_name = params.gex_reference_version ?: "gex_reference_version" CELLRANGER_MKREF( ch_fasta, CELLRANGER_MKGTF.out.gtf, - "gex_reference" + reference_name ) ch_versions = ch_versions.mix(CELLRANGER_MKREF.out.versions) ch_cellranger_gex_index = CELLRANGER_MKREF.out.reference.ifEmpty { [] } diff --git a/tests/main_pipeline_cellrangermulti.nf.test b/tests/main_pipeline_cellrangermulti.nf.test index 2d6e6a4a..62d897ee 100644 --- a/tests/main_pipeline_cellrangermulti.nf.test +++ b/tests/main_pipeline_cellrangermulti.nf.test @@ -15,6 +15,7 @@ nextflow_pipeline { input = "${baseDir}/assets/cellrangermulti_samplesheet.csv" cellranger_multi_barcodes = "${baseDir}/assets/cellranger_barcodes_samplesheet.csv" gex_frna_probe_set = "${baseDir}/assets/frna_probeset_subset.csv" + gex_reference_version = "GRCh38" fb_reference = "${baseDir}/assets/fb_reference.csv" fasta = 'https://ftp.ensembl.org/pub/release-110/fasta/homo_sapiens/dna/Homo_sapiens.GRCh38.dna.chromosome.14.fa.gz' gtf = 'https://ftp.ensembl.org/pub/release-110/gtf/homo_sapiens/Homo_sapiens.GRCh38.110.gtf.gz' @@ -83,5 +84,61 @@ nextflow_pipeline { } } + test("test-dataset_cellrangermulti_missing_gex_reference") { + + when { + params { + aligner = 'cellrangermulti' + outdir = "${outputDir}/results_cellrangermulti_missing_ref" + input = "${baseDir}/assets/cellrangermulti_samplesheet.csv" + cellranger_multi_barcodes = "${baseDir}/assets/cellranger_barcodes_samplesheet.csv" + gex_frna_probe_set = "${baseDir}/assets/frna_probeset_subset.csv" + fb_reference = "${baseDir}/assets/fb_reference.csv" + fasta = 'https://ftp.ensembl.org/pub/release-110/fasta/homo_sapiens/dna/Homo_sapiens.GRCh38.dna.chromosome.14.fa.gz' + gtf = 'https://ftp.ensembl.org/pub/release-110/gtf/homo_sapiens/Homo_sapiens.GRCh38.110.gtf.gz' + protocol = 'auto' + skip_cellbender = true + // Note: gex_reference_version is intentionally NOT provided to test validation + } + } + + then { + // Should fail with validation error + assertAll( + {assert !workflow.success}, + {assert workflow.failed}, + {assert workflow.stdout.any { it.contains("Parameter 'gex_reference_version' is required when 'gex_frna_probe_set' is provided and 'cellranger_index' is not provided") } } + ) + } + } + + test("test-dataset_cellrangermulti_mismatched_gex_reference") { + + when { + params { + aligner = 'cellrangermulti' + outdir = "${outputDir}/results_cellrangermulti_mismatch" + input = "${baseDir}/assets/cellrangermulti_samplesheet.csv" + cellranger_multi_barcodes = "${baseDir}/assets/cellranger_barcodes_samplesheet.csv" + gex_frna_probe_set = "${baseDir}/assets/frna_probeset_subset.csv" + gex_reference_version = "GRCm39" // Intentionally wrong - probeset is GRCh38 + fb_reference = "${baseDir}/assets/fb_reference.csv" + fasta = 'https://ftp.ensembl.org/pub/release-110/fasta/homo_sapiens/dna/Homo_sapiens.GRCh38.dna.chromosome.14.fa.gz' + gtf = 'https://ftp.ensembl.org/pub/release-110/gtf/homo_sapiens/Homo_sapiens.GRCh38.110.gtf.gz' + protocol = 'auto' + skip_cellbender = true + } + } + + then { + // Should fail with validation error + assertAll( + {assert !workflow.success}, + {assert workflow.failed}, + {assert workflow.stdout.any { it.contains("Parameter 'gex_reference_version' (GRCm39) does not match the probeset reference genome (GRCh38)") } } + ) + } + } + } diff --git a/tests/main_pipeline_cellrangermulti.nf.test.snap b/tests/main_pipeline_cellrangermulti.nf.test.snap index c0552368..050bd529 100644 --- a/tests/main_pipeline_cellrangermulti.nf.test.snap +++ b/tests/main_pipeline_cellrangermulti.nf.test.snap @@ -882,29 +882,29 @@ "cellrangermulti/mkgtf", "cellrangermulti/mkgtf/Homo_sapiens.GRCh38.dna.chromosome.14_genes.filtered.gtf", "cellrangermulti/mkref", - "cellrangermulti/mkref/gex_reference", - "cellrangermulti/mkref/gex_reference/fasta", - "cellrangermulti/mkref/gex_reference/fasta/genome.fa", - "cellrangermulti/mkref/gex_reference/fasta/genome.fa.fai", - "cellrangermulti/mkref/gex_reference/genes", - "cellrangermulti/mkref/gex_reference/genes/genes.gtf.gz", - "cellrangermulti/mkref/gex_reference/reference.json", - "cellrangermulti/mkref/gex_reference/star", - "cellrangermulti/mkref/gex_reference/star/Genome", - "cellrangermulti/mkref/gex_reference/star/SA", - "cellrangermulti/mkref/gex_reference/star/SAindex", - "cellrangermulti/mkref/gex_reference/star/chrLength.txt", - "cellrangermulti/mkref/gex_reference/star/chrName.txt", - "cellrangermulti/mkref/gex_reference/star/chrNameLength.txt", - "cellrangermulti/mkref/gex_reference/star/chrStart.txt", - "cellrangermulti/mkref/gex_reference/star/exonGeTrInfo.tab", - "cellrangermulti/mkref/gex_reference/star/exonInfo.tab", - "cellrangermulti/mkref/gex_reference/star/geneInfo.tab", - "cellrangermulti/mkref/gex_reference/star/genomeParameters.txt", - "cellrangermulti/mkref/gex_reference/star/sjdbInfo.txt", - "cellrangermulti/mkref/gex_reference/star/sjdbList.fromGTF.out.tab", - "cellrangermulti/mkref/gex_reference/star/sjdbList.out.tab", - "cellrangermulti/mkref/gex_reference/star/transcriptInfo.tab", + "cellrangermulti/mkref/GRCh38", + "cellrangermulti/mkref/GRCh38/fasta", + "cellrangermulti/mkref/GRCh38/fasta/genome.fa", + "cellrangermulti/mkref/GRCh38/fasta/genome.fa.fai", + "cellrangermulti/mkref/GRCh38/genes", + "cellrangermulti/mkref/GRCh38/genes/genes.gtf.gz", + "cellrangermulti/mkref/GRCh38/reference.json", + "cellrangermulti/mkref/GRCh38/star", + "cellrangermulti/mkref/GRCh38/star/Genome", + "cellrangermulti/mkref/GRCh38/star/SA", + "cellrangermulti/mkref/GRCh38/star/SAindex", + "cellrangermulti/mkref/GRCh38/star/chrLength.txt", + "cellrangermulti/mkref/GRCh38/star/chrName.txt", + "cellrangermulti/mkref/GRCh38/star/chrNameLength.txt", + "cellrangermulti/mkref/GRCh38/star/chrStart.txt", + "cellrangermulti/mkref/GRCh38/star/exonGeTrInfo.tab", + "cellrangermulti/mkref/GRCh38/star/exonInfo.tab", + "cellrangermulti/mkref/GRCh38/star/geneInfo.tab", + "cellrangermulti/mkref/GRCh38/star/genomeParameters.txt", + "cellrangermulti/mkref/GRCh38/star/sjdbInfo.txt", + "cellrangermulti/mkref/GRCh38/star/sjdbList.fromGTF.out.tab", + "cellrangermulti/mkref/GRCh38/star/sjdbList.out.tab", + "cellrangermulti/mkref/GRCh38/star/transcriptInfo.tab", "cellrangermulti/mkref/versions.yml", "cellrangermulti/mkvdjref", "cellrangermulti/mkvdjref/vdj_reference", @@ -1180,54 +1180,54 @@ "transcriptInfo.tab:md5,a27c997fc57e0e26beebb57cde77d986", "versions.yml:md5,7f4adb1e901d6610e40f18a6b7c1dc89", "regions.fa:md5,b10393dbe8cc3a3a1ebb3636e7369fa2", - "4PLEX_HUMAN_raw_matrix.h5ad:md5,cbfdafa68aae75611738fa9210b03f7d", - "4PLEX_HUMAN_raw_matrix.sce.rds:md5,084ac812ebb69ca3152abd2c7226d739", - "4PLEX_HUMAN_raw_matrix.seurat.rds:md5,acc22e948a2250907897f79e008ff3ea", - "Colorectal_BC3_filtered_matrix.h5ad:md5,a1de01a2e94b0829779d8f1d52465ff7", - "Colorectal_BC3_filtered_matrix.sce.rds:md5,91bf1e7978c8a0601230a5d37c50e402", - "Colorectal_BC3_filtered_matrix.seurat.rds:md5,ba8256b3a51477018f9c1e4e1aeec336", - "Colorectal_BC3_raw_matrix.h5ad:md5,9aa954fe00a6b413a67ac4f7f8d478dc", - "Colorectal_BC3_raw_matrix.sce.rds:md5,b00d8244f06eef3e5d0fae39c9e14196", - "Colorectal_BC3_raw_matrix.seurat.rds:md5,0d6a6222daf2b03cba426cb80b86914c", - "Liver_BC1_filtered_matrix.h5ad:md5,aecfe09a1cbd9f87161824be609db2de", - "Liver_BC1_filtered_matrix.sce.rds:md5,3f40a160ca908672593106cdceda3dc1", - "Liver_BC1_filtered_matrix.seurat.rds:md5,ce4a4c9cbd490e867771416b4ce6f458", - "Liver_BC1_raw_matrix.h5ad:md5,cd134ba9ecd79b11fc3f0d6bbd027bac", - "Liver_BC1_raw_matrix.sce.rds:md5,4806e7f7e00af77c9eb0e2666cedc96c", - 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"cellrangermulti/mkref/gex_reference/star/chrName.txt", - "cellrangermulti/mkref/gex_reference/star/chrNameLength.txt", - "cellrangermulti/mkref/gex_reference/star/chrStart.txt", - "cellrangermulti/mkref/gex_reference/star/exonGeTrInfo.tab", - "cellrangermulti/mkref/gex_reference/star/exonInfo.tab", - "cellrangermulti/mkref/gex_reference/star/geneInfo.tab", - "cellrangermulti/mkref/gex_reference/star/genomeParameters.txt", - "cellrangermulti/mkref/gex_reference/star/sjdbInfo.txt", - "cellrangermulti/mkref/gex_reference/star/sjdbList.fromGTF.out.tab", - "cellrangermulti/mkref/gex_reference/star/sjdbList.out.tab", - "cellrangermulti/mkref/gex_reference/star/transcriptInfo.tab", + "cellrangermulti/mkref/gex_reference_version", + "cellrangermulti/mkref/gex_reference_version/fasta", + "cellrangermulti/mkref/gex_reference_version/fasta/genome.fa", + "cellrangermulti/mkref/gex_reference_version/fasta/genome.fa.fai", + "cellrangermulti/mkref/gex_reference_version/genes", + "cellrangermulti/mkref/gex_reference_version/genes/genes.gtf.gz", + "cellrangermulti/mkref/gex_reference_version/reference.json", + "cellrangermulti/mkref/gex_reference_version/star", + "cellrangermulti/mkref/gex_reference_version/star/Genome", + "cellrangermulti/mkref/gex_reference_version/star/SA", + "cellrangermulti/mkref/gex_reference_version/star/SAindex", + "cellrangermulti/mkref/gex_reference_version/star/chrLength.txt", + "cellrangermulti/mkref/gex_reference_version/star/chrName.txt", + "cellrangermulti/mkref/gex_reference_version/star/chrNameLength.txt", + "cellrangermulti/mkref/gex_reference_version/star/chrStart.txt", + "cellrangermulti/mkref/gex_reference_version/star/exonGeTrInfo.tab", + "cellrangermulti/mkref/gex_reference_version/star/exonInfo.tab", + "cellrangermulti/mkref/gex_reference_version/star/geneInfo.tab", + "cellrangermulti/mkref/gex_reference_version/star/genomeParameters.txt", + "cellrangermulti/mkref/gex_reference_version/star/sjdbInfo.txt", + "cellrangermulti/mkref/gex_reference_version/star/sjdbList.fromGTF.out.tab", + "cellrangermulti/mkref/gex_reference_version/star/sjdbList.out.tab", + "cellrangermulti/mkref/gex_reference_version/star/transcriptInfo.tab", "cellrangermulti/mkref/versions.yml", "cellrangermulti/mkvdjref", "cellrangermulti/mkvdjref/vdj_reference", @@ -1807,33 +1807,33 @@ "transcriptInfo.tab:md5,213158051b61081ed7bd8f39c0324072", "versions.yml:md5,7f4adb1e901d6610e40f18a6b7c1dc89", "regions.fa:md5,be846142763972b29bbf65ff7e1406ee", - 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