Correct antigen analysis in cellranger multi (nf-core#4430)

* fix incomplete specification of BEAM antigens/controls

* update module tests for cellranger multi

* bugfix in process selector

* Remove environment.yml

---------

Co-authored-by: Gregor Sturm <mail@gregor-sturm.de>
Co-authored-by: Gregor Sturm <gregor.sturm@boehringer-ingelheim.com>

modules/nf-core/cellranger/multi/main.nf

@@ -18,7 +18,8 @@ process CELLRANGER_MULTI {
     path vdj_reference         , stageAs: "references/vdj/*"
     path vdj_primer_index      , stageAs: "references/vdj/primers/*"
     path fb_reference          , stageAs: "references/fb/*"
-    path beam_panel            , stageAs: "references/beam/panel/*"
+    path beam_antigen_panel    , stageAs: "references/beam/panel/antigens/*"
+    path beam_control_panel    , stageAs: "references/beam/panel/controls/*"
     path cmo_reference         , stageAs: "references/cmo/*"
     path cmo_barcodes          , stageAs: "references/cmo/barcodes/*"
     path cmo_barcode_assignment, stageAs: "references/cmo/sample_barcode_assignment/*"
@@ -45,20 +46,21 @@ process CELLRANGER_MULTI {
     // empty reference channels stage as "references"
     // empty FASTQ channels stage as "fastqs"
     // empty files stage as the file name, we check against 'EMPTY'
-    def gex_reference_name     = gex_reference.getName() != 'references'     ? gex_reference.getName()          : ''
-    def gex_frna_probeset_name = gex_frna_probeset.getBaseName() != 'EMPTY'  ? gex_frna_probeset.getName()      : ''
-    def gex_targetpanel_name   = gex_targetpanel.getBaseName() != 'EMPTY'    ? gex_targetpanel.getName()        : ''
-    def fb_reference_name      = fb_reference.getBaseName() != 'EMPTY'       ? fb_reference.getName()           : ''
-    def vdj_reference_name     = vdj_reference.getName() != 'references'     ? vdj_reference.getName()          : ''
-    def cmo_reference_name     = cmo_reference.getName() != 'EMPTY'          ? cmo_reference.getName()          : ''
-    def cmo_sample_assignment  = cmo_barcode_assignment.getName() != 'EMPTY' ? cmo_barcode_assignment.getName() : ''
-
-    def include_gex  = gex_fastqs.first().getName() != 'fastqs' && gex_reference ? '[gene-expression]'     : ''
-    def include_vdj  = vdj_fastqs.first().getName() != 'fastqs' && vdj_reference ? '[vdj]'                 : ''
-    def include_beam = beam_fastqs.first().getName() != 'fastqs' && beam_panel   ? '[antigen-specificity]' : ''
-    def include_cmo  = cmo_fastqs.first().getName() != 'fastqs' && cmo_barcodes  ? '[samples]'             : ''
-    def include_fb   = fb_reference.first().getName() != 'references'            ? '[feature]'             : ''
-    def include_frna = gex_frna_probeset_name && frna_sampleinfo                 ? '[samples]'             : ''
+    def gex_reference_name      = gex_reference.getName() != 'references'      ? gex_reference.getName()           : ''
+    def gex_frna_probeset_name  = gex_frna_probeset.getBaseName() != 'EMPTY'   ? gex_frna_probeset.getName()       : ''
+    def gex_targetpanel_name    = gex_targetpanel.getBaseName() != 'EMPTY'     ? gex_targetpanel.getName()         : ''
+    def fb_reference_name       = fb_reference.getBaseName() != 'EMPTY'        ? fb_reference.getName()            : ''
+    def vdj_reference_name      = vdj_reference.getName() != 'references'      ? vdj_reference.getName()           : ''
+    def cmo_reference_name      = cmo_reference.getName() != 'EMPTY'           ? cmo_reference.getName()           : ''
+    def cmo_sample_assignment   = cmo_barcode_assignment.getName() != 'EMPTY'  ? cmo_barcode_assignment.getName()  : ''
+    def beam_antigen_panel_name = beam_antigen_panel.getName() != 'EMPTY'      ? beam_antigen_panel.getName()      : ''
+
+    def include_gex  = gex_fastqs.first().getName() != 'fastqs' && gex_reference         ? '[gene-expression]'     : ''
+    def include_vdj  = vdj_fastqs.first().getName() != 'fastqs' && vdj_reference         ? '[vdj]'                 : ''
+    def include_beam = beam_fastqs.first().getName() != 'fastqs' && beam_control_panel   ? '[antigen-specificity]' : ''
+    def include_cmo  = cmo_fastqs.first().getName() != 'fastqs' && cmo_barcodes          ? '[samples]'             : ''
+    def include_fb   = fb_reference.first().getName() != 'references'                    ? '[feature]'             : ''
+    def include_frna = gex_frna_probeset_name && frna_sampleinfo                         ? '[samples]'             : ''
 
     def gex_reference_path = include_gex ? "reference,\$PWD/${gex_reference_name}" : ''
     def fb_reference_path  = include_fb  ? "reference,\$PWD/${fb_reference_name}"  : ''
@@ -73,11 +75,14 @@ process CELLRANGER_MULTI {
     // VDJ inner primer set
     def primer_index = vdj_primer_index.getBaseName() != 'EMPTY' ? "inner-enrichment-primers,\$PWD/references/primers/${vdj_primer_index.getName()}" : ''
 
+    // BEAM antigen list, remember that this is a Feature Barcode file
+    def beam_antigen_csv = include_beam && beam_antigen_panel_name != '' ? "reference,\$PWD/$beam_antigen_panel_name" : ''
+
     // pull CSV text from these reference panels
     // these references get appended directly to config file
-    def beam_csv_text  = include_beam && beam_panel.size() > 0      ? beam_panel.text      : ''
-    def cmo_csv_text   = include_cmo  && cmo_barcodes.size() > 0    ? cmo_barcodes         : ''
-    def frna_csv_text  = include_frna && frna_sampleinfo.size() > 0 ? frna_sampleinfo.text : ''
+    def beam_csv_text  = include_beam && beam_control_panel.size() > 0 ? beam_control_panel   : ''
+    def cmo_csv_text   = include_cmo  && cmo_barcodes.size() > 0       ? cmo_barcodes         : ''
+    def frna_csv_text  = include_frna && frna_sampleinfo.size() > 0    ? frna_sampleinfo      : ''
 
     // the feature barcodes section get options for either CRISPR or antibody capture assays
     def fb_options     = meta_ab?.options ? meta_ab.options : (meta_crispr?.options ? meta_crispr.options : [] )
@@ -127,50 +132,52 @@ process CELLRANGER_MULTI {
 
     """
     cat <<-CONFIG > $config
-        $include_gex
-        $gex_reference_path
-        $frna_probeset
-        $gex_options_filter_probes
-        $gex_options_r1_length
-        $gex_options_r2_length
-        $gex_options_chemistry
-        $gex_options_expect_cells
-        $gex_options_force_cells
-        $gex_options_no_secondary
-        $gex_options_no_bam
-        $gex_options_check_library_compatibility
-        $target_panel
-        $gex_options_no_target_umi_filter
-        $gex_options_include_introns
-        $cmo_options_min_assignment_confidence
-        $cmo_reference_path
-        $cmo_barcode_path
-
-        $include_fb
-        $fb_reference_path
-        $fb_options_r1_length
-        $fb_options_r2_length
-
-        $include_vdj
-        $vdj_reference_path
-        $primer_index
-        $vdj_options_r1_length
-        $vdj_options_r2_length
-
-        [libraries]
-        fastq_id,fastqs,lanes,feature_types
-        $fastq_gex
-        $fastq_vdj
-        $fastq_antibody
-        $fastq_beam
-        $fastq_crispr
-        $fastq_cmo
+    $include_gex
+    $gex_reference_path
+    $frna_probeset
+    $gex_options_filter_probes
+    $gex_options_r1_length
+    $gex_options_r2_length
+    $gex_options_chemistry
+    $gex_options_expect_cells
+    $gex_options_force_cells
+    $gex_options_no_secondary
+    $gex_options_no_bam
+    $gex_options_check_library_compatibility
+    $target_panel
+    $gex_options_no_target_umi_filter
+    $gex_options_include_introns
+    $cmo_options_min_assignment_confidence
+    $cmo_reference_path
+    $cmo_barcode_path
+
+    $include_fb
+    $fb_reference_path
+    $fb_options_r1_length
+    $fb_options_r2_length
+
+    $include_vdj
+    $vdj_reference_path
+    $primer_index
+    $vdj_options_r1_length
+    $vdj_options_r2_length
+
+    [libraries]
+    fastq_id,fastqs,lanes,feature_types
+    $fastq_gex
+    $fastq_vdj
+    $fastq_antibody
+    $fastq_beam
+    $fastq_crispr
+    $fastq_cmo
     CONFIG
 
     if [[ "$include_cmo" ]]; then echo "$include_cmo" >> $config; fi
     if [[ "$include_cmo" ]]; then cat $cmo_barcodes >> $config; fi
     if [[ "$include_beam" ]]; then echo "$include_beam" >> $config; fi
     if [[ "$include_beam" ]]; then cat "$beam_csv_text" >> $config; fi
+    if [[ "$include_beam" ]]; then echo "[feature]" >> $config; fi
+    if [[ "$include_beam" ]]; then echo "$beam_antigen_csv" >> $config; fi
     if [[ "$include_frna" ]]; then echo "$include_frna" >> $config; fi
     if [[ "$include_frna" ]]; then cat "$frna_csv_text" >> $config; fi
 

modules/nf-core/cellranger/multi/meta.yml

@@ -16,18 +16,86 @@ tools:
       homepage: https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/what-is-cell-ranger
       documentation: https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/using/tutorial_cp
       tool_dev_url: https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/using/tutorial_cp
-      licence: 10X Genomics EULA
+      licence: [10X Genomics EULA]
 input:
   - meta:
       type: map
       description: |
         Groovy Map containing sample information
         e.g. [ id:'test', single_end:false ]
-  - csv:
+  - gex_fastqs:
       type: file
-      description: Cell Ranger config file, in modified CSV format
+      description: FASTQ files for gene expression analysis
+      pattern: "*.fastq.gz"
+  - vdj_fastqs:
+      type: file
+      description: FASTQ files for V(D)J immunoprofiling analysis
+      pattern: "*.fastq.gz"
+  - ab_fastqs:
+      type: file
+      description: FASTQ files for antibody analysis
+      pattern: "*.fastq.gz"
+  - beam_fastqs:
+      type: file
+      description: FASTQ files for Barcode-Enabled Antigen Mapping (antigen analysis)
+      pattern: "*.fastq.gz"
+  - cmo_fastqs:
+      type: file
+      description: FASTQ files for cell multiplexing data
+      pattern: "*.fastq.gz"
+  - gex_reference:
+      type: directory
+      description: Folder containing Cellranger gene expression reference. Can also be a gzipped tarball
+      pattern: "*.tar.gz"
+  - gex_frna_probeset:
+      type: file
+      description: Fixed RNA profiling information containing custom probes in CSV format
+      pattern: "*.csv"
+  - gex_targetpanel:
+      type: file
+      description: Declaration of the target panel for Targeted Gene Expression analysis
+      pattern: "*.csv"
+  - vdj_reference:
+      type: directory
+      description: Folder containing Cellranger V(D)J reference. Can also be a gzipped tarball
+      pattern: "*.tar.gz"
+  - vdj_primer_index:
+      type: file
+      description: List of custom V(D)J inner enrichment primers
+      pattern: "*.csv"
+  - fb_reference:
+      type: file
+      description: The Feature Barcodes used for reference in Feature Barcoding Analysis
+      pattern: "*.csv"
+  - beam_antigen_panel:
+      type: file
+      description: The BEAM manifest in Feature Barcode CSV format
+      pattern: "*.csv"
+  - beam_control_panel:
+      type: file
+      description: The BEAM antigens set to control status, with corresponding MHC alleles, in Feature Barcode CSV format
+      pattern: "*.csv"
+  - cmo_reference:
+      type: file
+      description: Path to a custom Cell Multiplexing CSV reference IDs, or the `cmo-set` option in Cellranger
+      pattern: "*.csv"
+  - cmo_barcodes:
+      type: file
+      description: A CSV file appended to the Cellranger multi config linking samples to CMO IDs
+      pattern: "*.csv"
+  - cmo_barcode_assignment:
+      type: file
+      description: A CSV file that specifies the barcode-sample assignment in Cell Multiplexing analysis
+      pattern: "*.csv"
+  - frna_sampleinfo:
+      type: file
+      description: Sample information for fixed RNA analysis
       pattern: "*.csv"
 output:
+  - config:
+      type: file
+      description: The resolved Cellranger multi config used for analysis
+      pattern: "cellranger_multi_config.csv"
   - outs:
       type: file
       description: Files containing the outputs of Cell Ranger

tests/modules/nf-core/cellranger/multi/main.nf

@@ -161,7 +161,8 @@ ch_gex_frna_probeset     = Channel.fromPath( empty_file )
 ch_gex_targetpanel       = Channel.fromPath( empty_file )
 ch_vdj_primer_index      = Channel.fromPath( empty_file )
 ch_fb_reference          = Channel.fromPath( empty_file )
-ch_beam_panel            = Channel.fromPath( empty_file )
+ch_beam_antigen_panel    = Channel.fromPath( empty_file )
+ch_beam_control_panel    = Channel.fromPath( empty_file )
 ch_cmo_reference         = Channel.fromPath( empty_file )
 ch_cmo_barcodes          = Channel.fromPath( empty_file )
 ch_cmo_sample_assignment = Channel.fromPath( empty_file )
@@ -207,7 +208,8 @@ workflow test_cellranger_multi_10k_pbmc {
         ch_vdj_reference,
         ch_vdj_primer_index,
         ch_ab_reference_10k_pbmc,
-        ch_beam_panel,
+        ch_beam_antigen_panel,
+        ch_beam_control_panel,
         ch_cmo_reference,
         ch_cmo_barcodes,
         ch_cmo_sample_assignment,
@@ -257,7 +259,8 @@ workflow test_cellranger_multi_10k_pbmc_cmo {
         ch_vdj_ref_empty,
         ch_vdj_primer_index,
         ch_fb_reference,
-        ch_beam_panel,
+        ch_beam_antigen_panel,
+        ch_beam_control_panel,
         ch_cmo_reference_10k_pbmc_cmo,
         ch_cmo_barcodes_10k_pbmc_cmo,
         ch_cmo_sample_assignment,
@@ -304,7 +307,8 @@ workflow test_cellranger_multi_5k_cmvpos_tcells {
         ch_vdj_reference,
         ch_vdj_primer_index,
         ch_fb_reference_5k_cmvpos_tcells,
-        ch_beam_panel,
+        ch_beam_antigen_panel,
+        ch_beam_control_panel,
         ch_cmo_reference,
         ch_cmo_barcodes,
         ch_cmo_sample_assignment,

tests/modules/nf-core/cellranger/multi/nextflow.config

@@ -10,7 +10,7 @@ process {
         stageInMode = 'copy'
     }
 
-    withName: 'test_cellranger_multi_10k_5k_cmvpos_tcells:CELLRANGER_MULTI' {
+    withName: 'test_cellranger_multi_5k_cmvpos_tcells:CELLRANGER_MULTI' {
         stageInMode = 'copy'
     }
 }

tests/modules/nf-core/cellranger/multi/test.yml

@@ -8,15 +8,14 @@
       contains:
         - "[gene-expression]"
     - path: output/cellranger/genome.filtered.gtf
-      md5sum: 428eb76f23f848bbb727fa5001a4d0fb
     - path: output/cellranger/homo_sapiens_chr22_reference/fasta/genome.fa
       md5sum: f315020d899597c1b57e5fe9f60f4c3e
     - path: output/cellranger/homo_sapiens_chr22_reference/fasta/genome.fa.fai
       md5sum: 3520cd30e1b100e55f578db9c855f685
     - path: output/cellranger/homo_sapiens_chr22_reference/genes/genes.gtf.gz
-      md5sum: 6d9b5f409bfea95022bc25b9590e194e
     - path: output/cellranger/homo_sapiens_chr22_reference/reference.json
-      md5sum: 55de930f10bc6b116c3f0c0fe0cbf4fc
+      contains:
+        - "homo_sapiens_chr22_reference"
     - path: output/cellranger/homo_sapiens_chr22_reference/star/Genome
       md5sum: 22102926fadf5890e905ca71b2da3f35
     - path: output/cellranger/homo_sapiens_chr22_reference/star/SA
@@ -32,7 +31,6 @@
     - path: output/cellranger/homo_sapiens_chr22_reference/star/chrStart.txt
       md5sum: bc73df776dd3d5bb9cfcbcba60880519
     - path: output/cellranger/homo_sapiens_chr22_reference/star/exonGeTrInfo.tab
-      md5sum: d04497f69d6ef889efd4d34fe63edcc4
     - path: output/cellranger/homo_sapiens_chr22_reference/star/exonInfo.tab
       md5sum: 0d560290fab688b7268d88d5494bf9fe
     - path: output/cellranger/homo_sapiens_chr22_reference/star/geneInfo.tab
@@ -123,15 +121,16 @@
       contains:
         - "[gene-expression]"
     - path: output/cellranger/genome.filtered.gtf
-      md5sum: 428eb76f23f848bbb727fa5001a4d0fb
+      contains:
+        - "chr22"
     - path: output/cellranger/homo_sapiens_chr22_reference/fasta/genome.fa
       md5sum: f315020d899597c1b57e5fe9f60f4c3e
     - path: output/cellranger/homo_sapiens_chr22_reference/fasta/genome.fa.fai
       md5sum: 3520cd30e1b100e55f578db9c855f685
     - path: output/cellranger/homo_sapiens_chr22_reference/genes/genes.gtf.gz
-      md5sum: 6d9b5f409bfea95022bc25b9590e194e
     - path: output/cellranger/homo_sapiens_chr22_reference/reference.json
-      md5sum: 55de930f10bc6b116c3f0c0fe0cbf4fc
+      contains:
+        - "homo_sapiens_chr22_reference"
     - path: output/cellranger/homo_sapiens_chr22_reference/star/Genome
       md5sum: 22102926fadf5890e905ca71b2da3f35
     - path: output/cellranger/homo_sapiens_chr22_reference/star/SA
@@ -147,7 +146,6 @@
     - path: output/cellranger/homo_sapiens_chr22_reference/star/chrStart.txt
       md5sum: bc73df776dd3d5bb9cfcbcba60880519
     - path: output/cellranger/homo_sapiens_chr22_reference/star/exonGeTrInfo.tab
-      md5sum: d04497f69d6ef889efd4d34fe63edcc4
     - path: output/cellranger/homo_sapiens_chr22_reference/star/exonInfo.tab
       md5sum: 0d560290fab688b7268d88d5494bf9fe
     - path: output/cellranger/homo_sapiens_chr22_reference/star/geneInfo.tab
@@ -225,15 +223,16 @@
       contains:
         - "[gene-expression]"
     - path: output/cellranger/genome.filtered.gtf
-      md5sum: 428eb76f23f848bbb727fa5001a4d0fb
+      contains:
+        - "chr22"
     - path: output/cellranger/homo_sapiens_chr22_reference/fasta/genome.fa
       md5sum: f315020d899597c1b57e5fe9f60f4c3e
     - path: output/cellranger/homo_sapiens_chr22_reference/fasta/genome.fa.fai
       md5sum: 3520cd30e1b100e55f578db9c855f685
     - path: output/cellranger/homo_sapiens_chr22_reference/genes/genes.gtf.gz
-      md5sum: 6d9b5f409bfea95022bc25b9590e194e
     - path: output/cellranger/homo_sapiens_chr22_reference/reference.json
-      md5sum: 55de930f10bc6b116c3f0c0fe0cbf4fc
+      contains:
+        - "homo_sapiens_chr22_reference"
     - path: output/cellranger/homo_sapiens_chr22_reference/star/Genome
       md5sum: 22102926fadf5890e905ca71b2da3f35
     - path: output/cellranger/homo_sapiens_chr22_reference/star/SA
@@ -249,7 +248,6 @@
     - path: output/cellranger/homo_sapiens_chr22_reference/star/chrStart.txt
       md5sum: bc73df776dd3d5bb9cfcbcba60880519
     - path: output/cellranger/homo_sapiens_chr22_reference/star/exonGeTrInfo.tab
-      md5sum: d04497f69d6ef889efd4d34fe63edcc4
     - path: output/cellranger/homo_sapiens_chr22_reference/star/exonInfo.tab
       md5sum: 0d560290fab688b7268d88d5494bf9fe
     - path: output/cellranger/homo_sapiens_chr22_reference/star/geneInfo.tab