From 95321c8caec3343f3d167a9d427bc0be55fee6a4 Mon Sep 17 00:00:00 2001
From: Matteo Bonfanti <matteo.bonfanti@fht.org>
Date: Mon, 20 Oct 2025 17:22:06 +0200
Subject: [PATCH 01/18] implementation of spaceranger

---
 assets/samplesheet_test.csv                   |   6 +-
 assets/schema_input.json                      |  42 ++++
 conf/modules.config                           |  43 ++++
 conf/test.config                              |   8 +-
 main.nf                                       |   4 +-
 modules.json                                  |  10 +
 modules/nf-core/spaceranger/count/main.nf     |  76 ++++++
 modules/nf-core/spaceranger/count/meta.yml    | 119 +++++++++
 .../spaceranger/count/tests/main.nf.test      | 228 ++++++++++++++++++
 .../spaceranger/count/tests/main.nf.test.snap |  90 +++++++
 .../spaceranger/count/tests/nextflow.config   |   5 +
 modules/nf-core/spaceranger/mkgtf/main.nf     |  51 ++++
 modules/nf-core/spaceranger/mkgtf/meta.yml    |  47 ++++
 .../spaceranger/mkgtf/tests/main.nf.test      |  59 +++++
 .../spaceranger/mkgtf/tests/main.nf.test.snap |  29 +++
 subworkflows/local/prepare_ref/main.nf        |  28 ++-
 workflows/spatialomics.nf                     |  29 ++-
 17 files changed, 862 insertions(+), 12 deletions(-)
 create mode 100644 modules/nf-core/spaceranger/count/main.nf
 create mode 100644 modules/nf-core/spaceranger/count/meta.yml
 create mode 100644 modules/nf-core/spaceranger/count/tests/main.nf.test
 create mode 100644 modules/nf-core/spaceranger/count/tests/main.nf.test.snap
 create mode 100644 modules/nf-core/spaceranger/count/tests/nextflow.config
 create mode 100644 modules/nf-core/spaceranger/mkgtf/main.nf
 create mode 100644 modules/nf-core/spaceranger/mkgtf/meta.yml
 create mode 100644 modules/nf-core/spaceranger/mkgtf/tests/main.nf.test
 create mode 100644 modules/nf-core/spaceranger/mkgtf/tests/main.nf.test.snap

diff --git a/assets/samplesheet_test.csv b/assets/samplesheet_test.csv
index 6c5eb8a..4aa0b09 100644
--- a/assets/samplesheet_test.csv
+++ b/assets/samplesheet_test.csv
@@ -1,3 +1,3 @@
-sample,fastq_1,fastq_2
-Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2,https://raw.githubusercontent.com/nf-core/test-datasets/spatialvi/testdata/human-lung-cancer-post-xenium_hd_ffpe/Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2_S1_L001_R1_001.fastq.gz,https://raw.githubusercontent.com/nf-core/test-datasets/spatialvi/testdata/human-lung-cancer-post-xenium_hd_ffpe/Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2_S1_L001_R2_001.fastq.gz
-Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2,https://raw.githubusercontent.com/nf-core/test-datasets/spatialvi/testdata/human-lung-cancer-post-xenium_hd_ffpe/Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2_S1_L002_R1_001.fastq.gz,https://raw.githubusercontent.com/nf-core/test-datasets/spatialvi/testdata/human-lung-cancer-post-xenium_hd_ffpe/Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2_S1_L002_R2_001.fastq.gz
+sample,fastq_1,fastq_2,image,cytaimage,slide,area,darkimage,colorizedimage,manual_alignment,slidefile
+Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2,https://raw.githubusercontent.com/nf-core/test-datasets/spatialvi/testdata/human-lung-cancer-post-xenium_hd_ffpe/Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2_S1_L001_R1_001.fastq.gz,https://raw.githubusercontent.com/nf-core/test-datasets/spatialvi/testdata/human-lung-cancer-post-xenium_hd_ffpe/Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2_S1_L001_R2_001.fastq.gz,https://raw.githubusercontent.com/nf-core/test-datasets/spatialvi/testdata/human-lung-cancer-post-xenium_hd_ffpe/Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2_tissue_image.btf,https://raw.githubusercontent.com/nf-core/test-datasets/spatialvi/testdata/human-lung-cancer-post-xenium_hd_ffpe/Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2_image.tif,H1-84QJZFR,D1,,,,,
+Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2,https://raw.githubusercontent.com/nf-core/test-datasets/spatialvi/testdata/human-lung-cancer-post-xenium_hd_ffpe/Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2_S1_L002_R1_001.fastq.gz,https://raw.githubusercontent.com/nf-core/test-datasets/spatialvi/testdata/human-lung-cancer-post-xenium_hd_ffpe/Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2_S1_L002_R2_001.fastq.gz,https://raw.githubusercontent.com/nf-core/test-datasets/spatialvi/testdata/human-lung-cancer-post-xenium_hd_ffpe/Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2_tissue_image.btf,https://raw.githubusercontent.com/nf-core/test-datasets/spatialvi/testdata/human-lung-cancer-post-xenium_hd_ffpe/Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2_image.tif,H1-84QJZFR,D1,,,,,
diff --git a/assets/schema_input.json b/assets/schema_input.json
index 4df003e..83ae24a 100644
--- a/assets/schema_input.json
+++ b/assets/schema_input.json
@@ -26,6 +26,48 @@
                 "exists": true,
                 "pattern": "^([\\S\\s]*\\/)?[^\\s\\/]+\\.f(ast)?q\\.gz$",
                 "errorMessage": "FastQ file for reads 2 cannot contain spaces and must have extension '.fq.gz' or '.fastq.gz'"
+            },
+            "image": {
+                "type": "string",
+                "format": "file-path",
+                "exists": true,
+                "meta": ["image"]
+            },
+            "cytaimage": {
+                "type": "string",
+                "format": "file-path",
+                "exists": true,
+                "meta": ["cytaimage"]
+            },
+            "slide": {
+                "type": "string",
+                "meta": ["slide"]
+            },
+            "area": {
+                "type": "string",
+                "meta": ["area"]
+            },
+            "darkimage": {
+                "type": "string",
+                "format": "file-path",
+                "exists": true,
+                "meta": ["darkimage"]
+            },
+            "colorizedimage": {
+                "type": "string",
+                "format": "file-path",
+                "exists": true,
+                "meta": ["colorizedimage"]
+            },
+            "manual_alignment": {
+                "type": "string",
+                "meta": ["alignment"]
+            },
+            "slidefile": {
+                "type": "string",
+                "format": "file-path",
+                "exists": true,
+                "meta": ["slidefile"]
             }
         },
         "required": ["sample", "fastq_1"]
diff --git a/conf/modules.config b/conf/modules.config
index d203d2b..97c2546 100644
--- a/conf/modules.config
+++ b/conf/modules.config
@@ -22,6 +22,49 @@ process {
         ext.args = '--quiet'
     }
 
+    withName: SPACERANGER_MKGTF {
+        publishDir = [
+            path: "${params.outdir}/reference/mkgtf",
+            mode: params.publish_dir_mode,
+            saveAs: { filename -> filename.equals('versions.yml') ? null : filename }
+        ]
+        ext.args = "--attribute=gene_biotype:protein_coding \
+                    --attribute=gene_biotype:lincRNA \
+                    --attribute=gene_biotype:antisense \
+                    --attribute=gene_biotype:IG_LV_gene \
+                    --attribute=gene_biotype:IG_V_gene \
+                    --attribute=gene_biotype:IG_V_pseudogene \
+                    --attribute=gene_biotype:IG_D_gene \
+                    --attribute=gene_biotype:IG_J_gene \
+                    --attribute=gene_biotype:IG_J_pseudogene \
+                    --attribute=gene_biotype:IG_C_gene \
+                    --attribute=gene_biotype:IG_C_pseudogene \
+                    --attribute=gene_biotype:TR_V_gene \
+                    --attribute=gene_biotype:TR_V_pseudogene \
+                    --attribute=gene_biotype:TR_D_gene \
+                    --attribute=gene_biotype:TR_J_gene \
+                    --attribute=gene_biotype:TR_J_pseudogene \
+                    --attribute=gene_biotype:TR_C_gene"
+    }
+
+    withName: SPACERANGER_MKREF {
+        publishDir = [
+            path: "${params.outdir}/reference/mkref",
+            mode: params.publish_dir_mode,
+            saveAs: { filename -> filename.equals('versions.yml') ? null : filename }
+        ]
+    }
+
+    withName: SPACERANGER_COUNT {
+        publishDir = [
+            path: { "${params.outdir}/${meta.id}/count" },
+            mode: params.publish_dir_mode,
+            saveAs: { filename -> filename.equals('versions.yml') ? null : filename }
+        ]
+        ext.args   = "--create-bam false"
+        time   = { 240.h * task.attempt }
+    }
+
     withName: 'MULTIQC' {
         ext.args   = { params.multiqc_title ? "--title \"$params.multiqc_title\"" : '' }
         publishDir = [
diff --git a/conf/test.config b/conf/test.config
index 989bf3a..82c4d57 100644
--- a/conf/test.config
+++ b/conf/test.config
@@ -23,7 +23,9 @@ params {
     config_profile_description = 'Minimal test dataset to check pipeline function'
 
     // Input and output
-    input  = "${projectDir}/assets/samplesheet_test.csv"
-    fasta  = "https://raw.githubusercontent.com/nf-core/test-datasets/references/references/GRCh38_chr21/GRCh38_chr21.fa"
-    gtf    = "https://raw.githubusercontent.com/nf-core/test-datasets/references/references/GRCh38_chr21/GRCh38_chr21.gtf"
+    input             = "${projectDir}/assets/samplesheet_test.csv"
+    probeset          = "https://raw.githubusercontent.com/nf-core/test-datasets/spatialvi/testdata/human-lung-cancer-post-xenium_hd_ffpe/Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2_probe_set.csv"
+    spaceranger_index = "https://raw.githubusercontent.com/nf-core/test-datasets/spatialvi/testdata/GRCh38.tar.gz"
+
+
 }
diff --git a/main.nf b/main.nf
index 3cdb747..0624405 100644
--- a/main.nf
+++ b/main.nf
@@ -49,6 +49,7 @@ workflow NFDATAOMICS_SPATIALOMICS {
     ch_gtf                = params.gtf               ? Channel.value(file(params.gtf, checkIfExists: true))               : Channel.empty()
     ch_gff                = params.gff               ? Channel.value(file(params.gff, checkIfExists: true))               : Channel.empty()
     ch_spaceranger_index  = params.spaceranger_index ? Channel.value(file(params.spaceranger_index, checkIfExists: true)) : Channel.empty()
+    ch_probeset           = params.probeset          ? Channel.value(file(params.probeset, checkIfExists: true))          : Channel.empty()
 
     //
     // WORKFLOW: Run pipeline
@@ -58,7 +59,8 @@ workflow NFDATAOMICS_SPATIALOMICS {
         ch_fasta,
         ch_gtf,
         ch_gff,
-        ch_spaceranger_index
+        ch_spaceranger_index,
+        ch_probeset
     )
     emit:
     multiqc_report = SPATIALOMICS.out.multiqc_report // channel: /path/to/multiqc_report.html
diff --git a/modules.json b/modules.json
index 75fb70b..dbc41c1 100644
--- a/modules.json
+++ b/modules.json
@@ -35,6 +35,16 @@
                         "git_sha": "e10b76ca0c66213581bec2833e30d31f239dec0b",
                         "installed_by": ["modules"]
                     },
+                    "spaceranger/count": {
+                        "branch": "master",
+                        "git_sha": "41dfa3f7c0ffabb96a6a813fe321c6d1cc5b6e46",
+                        "installed_by": ["modules"]
+                    },
+                    "spaceranger/mkgtf": {
+                        "branch": "master",
+                        "git_sha": "41dfa3f7c0ffabb96a6a813fe321c6d1cc5b6e46",
+                        "installed_by": ["modules"]
+                    },
                     "spaceranger/mkref": {
                         "branch": "master",
                         "git_sha": "41dfa3f7c0ffabb96a6a813fe321c6d1cc5b6e46",
diff --git a/modules/nf-core/spaceranger/count/main.nf b/modules/nf-core/spaceranger/count/main.nf
new file mode 100644
index 0000000..2436210
--- /dev/null
+++ b/modules/nf-core/spaceranger/count/main.nf
@@ -0,0 +1,76 @@
+process SPACERANGER_COUNT {
+    tag "$meta.id"
+    label 'process_high'
+
+    container "nf-core/spaceranger:3.1.3"
+
+    input:
+    tuple val(meta), path("fastqs/${meta.id}_S1_L001_R?_001.fastq.gz")
+    tuple val(meta2), path(image), val(slide), val(area), path(cytaimage), path(darkimage), path(colorizedimage), path(alignment), path(slidefile)
+    path(reference)
+    path(probeset)
+
+    output:
+    tuple val(meta), path("outs/**"), emit: outs
+    path "versions.yml", emit: versions
+
+    when:
+    task.ext.when == null || task.ext.when
+
+    script:
+    // Exit if running this module with -profile conda / -profile mamba
+    if (workflow.profile.tokenize(',').intersect(['conda', 'mamba']).size() >= 1) {
+        error "SPACERANGER_COUNT module does not support Conda. Please use Docker / Singularity / Podman instead."
+    }
+    def args = task.ext.args ?: ''
+    def prefix = task.ext.prefix ?: "${meta.id}"
+    // Add flags for optional inputs on demand.
+    def probeset = probeset ? "--probe-set=\"${probeset}\"" : ""
+    def alignment = alignment ? "--loupe-alignment=\"${alignment}\"" : ""
+    def slidefile = slidefile ? "--slidefile=\"${slidefile}\"" : ""
+    def image = image ? "--image=\"${image}\"" : ""
+    def cytaimage = cytaimage ? "--cytaimage=\"${cytaimage}\"" : ""
+    def darkimage = darkimage ? "--darkimage=\"${darkimage}\"" : ""
+    def colorizedimage = colorizedimage ? "--colorizedimage=\"${colorizedimage}\"" : ""
+    if (slide.matches("visium-(.*)") && area == "" && slidefile == "") {
+        slide_and_area = "--unknown-slide=\"${slide}\""
+    } else {
+        slide_and_area = "--slide=\"${slide}\" --area=\"${area}\""
+    }
+    """
+    spaceranger count \\
+        --id="${prefix}" \\
+        --sample="${meta.id}" \\
+        --fastqs=fastqs \\
+        --transcriptome="${reference}" \\
+        --localcores=${task.cpus} \\
+        --localmem=${task.memory.toGiga()} \\
+        $image $cytaimage $darkimage $colorizedimage \\
+        $slide_and_area \\
+        $probeset \\
+        $alignment \\
+        $slidefile \\
+        $args
+    mv ${prefix}/outs outs
+
+    cat <<-END_VERSIONS > versions.yml
+    "${task.process}":
+        spaceranger: \$(spaceranger -V | sed -e "s/spaceranger spaceranger-//g")
+    END_VERSIONS
+    """
+
+    stub:
+    // Exit if running this module with -profile conda / -profile mamba
+    if (workflow.profile.tokenize(',').intersect(['conda', 'mamba']).size() >= 1) {
+        error "SPACERANGER_COUNT module does not support Conda. Please use Docker / Singularity / Podman instead."
+    }
+    """
+    mkdir -p outs/
+    touch outs/fake_file.txt
+
+    cat <<-END_VERSIONS > versions.yml
+    "${task.process}":
+        spaceranger: \$(spaceranger -V | sed -e "s/spaceranger spaceranger-//g")
+    END_VERSIONS
+    """
+}
diff --git a/modules/nf-core/spaceranger/count/meta.yml b/modules/nf-core/spaceranger/count/meta.yml
new file mode 100644
index 0000000..cf6efb5
--- /dev/null
+++ b/modules/nf-core/spaceranger/count/meta.yml
@@ -0,0 +1,119 @@
+# yaml-language-server: $schema=https://raw.githubusercontent.com/nf-core/modules/master/modules/yaml-schema.json
+name: "spaceranger_count"
+description: Module to use the 10x Space Ranger pipeline to process 10x spatial transcriptomics
+  data
+keywords:
+  - align
+  - count
+  - spatial
+  - spaceranger
+  - imaging
+tools:
+  - "spaceranger":
+      description: |
+        Visium Spatial Gene Expression is a next-generation molecular profiling solution for classifying tissue
+        based on total mRNA. Space Ranger is a set of analysis pipelines that process Visium Spatial Gene Expression
+        data with brightfield and fluorescence microscope images. Space Ranger allows users to map the whole
+        transcriptome in formalin fixed paraffin embedded (FFPE) and fresh frozen tissues to discover novel
+        insights into normal development, disease pathology, and clinical translational research. Space Ranger provides
+        pipelines for end to end analysis of Visium Spatial Gene Expression experiments.
+      homepage: "https://support.10xgenomics.com/spatial-gene-expression/software/pipelines/latest/what-is-space-ranger"
+      documentation: "https://support.10xgenomics.com/spatial-gene-expression/software/pipelines/latest/what-is-space-ranger"
+      tool_dev_url: "https://support.10xgenomics.com/spatial-gene-expression/software/pipelines/latest/what-is-space-ranger"
+      licence:
+        - "10x Genomics EULA"
+      identifier: ""
+input:
+  - - meta:
+        type: map
+        description: |
+          Groovy Map containing sample information
+          e.g. [ id:'test', slide:'10L13-020', area: 'B1']
+
+          `id`, `slide` and `area` are mandatory information!
+    - reads:
+        type: file
+        description: |
+          List of input FastQ files of size 1 and 2 for single-end and paired-end data,
+          respectively.
+        pattern: "${Sample_Name}_S1_L00${Lane_Number}_${I1,I2,R1,R2}_001.fastq.gz"
+        ontologies: []
+    - image:
+        type: file
+        description: Brightfield tissue H&E image in JPEG or TIFF format.
+        pattern: "*.{tif,tiff,jpg,jpeg}"
+        ontologies: []
+    - slide:
+        type: string
+        description: Visium slide ID used for the sample.
+    - area:
+        type: string
+        description: Visium slide capture area used for the sample.
+    - cytaimage:
+        type: file
+        description: |
+          CytAssist instrument captured eosin stained Brightfield tissue image with fiducial
+          frame in TIFF format. The size of this image is set at 3k in both dimensions and this image should
+          not be modified any way before passing it as input to either Space Ranger or Loupe Browser.
+        pattern: "*.{tif,tiff}"
+        ontologies: []
+    - darkimage:
+        type: file
+        description: |
+          Optional for dark background fluorescence microscope image input. Multi-channel, dark-background fluorescence
+          image as either a single, multi-layer TIFF file or as multiple TIFF or JPEG files.
+        pattern: "*.{tif,tiff,jpg,jpeg}"
+        ontologies: []
+    - colorizedimage:
+        type: file
+        description: |
+          Required for color composite fluorescence microscope image input.
+          A color composite of one or more fluorescence image channels saved as a single-page,
+          single-file color TIFF or JPEG.
+        pattern: "*.{tif,tiff,jpg,jpeg}"
+        ontologies: []
+    - alignment:
+        type: file
+        description: OPTIONAL - Path to manual image alignment.
+        pattern: "*.json"
+        ontologies:
+          - edam: http://edamontology.org/format_3464 # JSON
+    - slidefile:
+        type: file
+        description: OPTIONAL - Path to slide specifications.
+        pattern: "*.json"
+        ontologies:
+          - edam: http://edamontology.org/format_3464 # JSON
+  - reference:
+      type: directory
+      description: Folder containing all the reference indices needed by Space Ranger
+  - probeset:
+      type: file
+      description: OPTIONAL - Probe set specification.
+      pattern: "*.csv"
+      ontologies:
+        - edam: http://edamontology.org/format_3752 # CSV
+output:
+  outs:
+    - - meta:
+          type: map
+          description: |
+            Groovy Map containing sample information
+            e.g. [ id:'test', single_end:false ]
+      - outs/**:
+          type: file
+          description: Files containing the outputs of Space Ranger, see official 10X
+            Genomics documentation for a complete list
+          pattern: "outs/*"
+          ontologies: []
+  versions:
+    - versions.yml:
+        type: file
+        description: File containing software versions
+        pattern: "versions.yml"
+        ontologies:
+          - edam: http://edamontology.org/format_3750 # YAML
+authors:
+  - "@grst"
+maintainers:
+  - "@grst"
diff --git a/modules/nf-core/spaceranger/count/tests/main.nf.test b/modules/nf-core/spaceranger/count/tests/main.nf.test
new file mode 100644
index 0000000..3d65e68
--- /dev/null
+++ b/modules/nf-core/spaceranger/count/tests/main.nf.test
@@ -0,0 +1,228 @@
+nextflow_process {
+
+    name "Test Process SPACERANGER_COUNT"
+    script "../main.nf"
+    config "./nextflow.config"
+    process "SPACERANGER_COUNT"
+
+    tag "modules"
+    tag "modules_nfcore"
+    tag "spaceranger"
+    tag "spaceranger/count"
+    tag "spaceranger/mkgtf"
+    tag "spaceranger/mkref"
+
+    test("spaceranger v1 - homo_sapiens - fasta - gtf - fastq - tif - csv") {
+
+        setup {
+            run("SPACERANGER_MKGTF") {
+                script "../../mkgtf/main.nf"
+                process {
+                    """
+                    input[0] = file(params.modules_testdata_base_path + 'genomics/homo_sapiens/genome/genome.gtf', checkIfExists: true)
+                    """
+                }
+            }
+        }
+
+        setup {
+            run("SPACERANGER_MKREF") {
+                script "../../mkref/main.nf"
+                process {
+                    """
+                    input[0] = file(params.modules_testdata_base_path + 'genomics/homo_sapiens/genome/genome.fasta', checkIfExists: true)
+                    input[1] = file(params.modules_testdata_base_path + 'genomics/homo_sapiens/genome/genome.gtf', checkIfExists: true)
+                    input[2] = 'homo_sapiens_chr22_reference'
+                    """
+                }
+            }
+        }
+
+        when {
+            process {
+                """
+                input[0] = [
+                    [
+                        id: 'Visium_FFPE_Human_Ovarian_Cancer'
+                    ], // Meta map
+                    [
+                        file(params.modules_testdata_base_path + 'genomics/homo_sapiens/10xgenomics/spaceranger/human-ovarian-cancer-1-standard_v1_ffpe/Visium_FFPE_Human_Ovarian_Cancer_S1_L001_R1_001.fastq.gz', checkIfExists: true),
+                        file(params.modules_testdata_base_path + 'genomics/homo_sapiens/10xgenomics/spaceranger/human-ovarian-cancer-1-standard_v1_ffpe/Visium_FFPE_Human_Ovarian_Cancer_S1_L001_R2_001.fastq.gz', checkIfExists: true)
+                    ], // Reads
+                    file(params.modules_testdata_base_path + 'genomics/homo_sapiens/10xgenomics/spaceranger/human-ovarian-cancer-1-standard_v1_ffpe/Visium_FFPE_Human_Ovarian_Cancer_image.jpg', checkIfExists: true), // Image
+                    'V10L13-020', // Slide
+                    'D1', // Area
+                    [], // Cytaimage
+                    [], // Darkimage
+                    [], // Colorizedimage
+                    [], // Manual alignment (default: automatic alignment)
+                    [], // Slide specification (default: automatic download)
+                ]
+                input[1] = SPACERANGER_MKREF.out.reference // Reference
+                input[2] = [] // Probeset (default: use the one included with Space Ranger)
+                """
+            }
+        }
+
+        then {
+            assertAll(
+                { assert process.success },
+                { assert snapshot(
+                    process.out.versions,
+                    process.out.outs.get(0).get(1).findAll { file(it).name !in [
+                        'web_summary.html',
+                        'scalefactors_json.json',
+                        'barcodes.tsv.gz',
+                        'features.tsv.gz',
+                        'matrix.mtx.gz'
+                        ]}
+                    ).match()
+                },
+                { assert file(process.out.outs.get(0).get(1).find { file(it).name == 'web_summary.html' }).exists() },
+                { assert file(process.out.outs.get(0).get(1).find { file(it).name == 'scalefactors_json.json' }).exists() },
+                { assert file(process.out.outs.get(0).get(1).find { file(it).name == 'barcodes.tsv.gz' }).exists() },
+                { assert file(process.out.outs.get(0).get(1).find { file(it).name == 'features.tsv.gz' }).exists() },
+                { assert file(process.out.outs.get(0).get(1).find { file(it).name == 'matrix.mtx.gz' }).exists() }
+            )
+        }
+    }
+
+    test("spaceranger v1 (stub) - homo_sapiens - fasta - gtf - fastq - tif - csv") {
+
+        setup {
+            run("SPACERANGER_MKGTF") {
+                script "../../mkgtf/main.nf"
+                process {
+                    """
+                    input[0] = file(params.modules_testdata_base_path + 'genomics/homo_sapiens/genome/genome.gtf', checkIfExists: true)
+                    """
+                }
+            }
+        }
+
+        setup {
+            run("SPACERANGER_MKREF") {
+                script "../../mkref/main.nf"
+                process {
+                    """
+                    input[0] = file(params.modules_testdata_base_path + 'genomics/homo_sapiens/genome/genome.fasta', checkIfExists: true)
+                    input[1] = file(params.modules_testdata_base_path + 'genomics/homo_sapiens/genome/genome.gtf', checkIfExists: true)
+                    input[2] = 'homo_sapiens_chr22_reference'
+                    """
+                }
+            }
+        }
+
+        options "-stub"
+
+        when {
+            process {
+                """
+                input[0] = [
+                    [
+                        id: 'Visium_FFPE_Human_Ovarian_Cancer'
+                    ], // Meta map
+                    [
+                        file(params.modules_testdata_base_path + 'genomics/homo_sapiens/10xgenomics/spaceranger/human-ovarian-cancer-1-standard_v1_ffpe/Visium_FFPE_Human_Ovarian_Cancer_S1_L001_R1_001.fastq.gz', checkIfExists: true),
+                        file(params.modules_testdata_base_path + 'genomics/homo_sapiens/10xgenomics/spaceranger/human-ovarian-cancer-1-standard_v1_ffpe/Visium_FFPE_Human_Ovarian_Cancer_S1_L001_R2_001.fastq.gz', checkIfExists: true)
+                    ], // Reads
+                    file(params.modules_testdata_base_path + 'genomics/homo_sapiens/10xgenomics/spaceranger/human-ovarian-cancer-1-standard_v1_ffpe/Visium_FFPE_Human_Ovarian_Cancer_image.jpg', checkIfExists: true), // Image
+                    'V10L13-020', // Slide
+                    'D1', // Area
+                    [], // Cytaimage
+                    [], // Darkimage
+                    [], // Colorizedimage
+                    [], // Manual alignment (default: automatic alignment)
+                    [], // Slide specification (default: automatic download)
+                ]
+                input[1] = SPACERANGER_MKREF.out.reference // Reference
+                input[2] = [] // Probeset (default: use the one included with Space Ranger)
+                """
+            }
+        }
+
+        then {
+            assertAll(
+                { assert process.success },
+                { assert snapshot(process.out.versions).match() },
+            )
+        }
+    }
+
+    test("spaceranger v2 - homo_sapiens - fasta - gtf - fastq - tif - csv") {
+        setup {
+            run("SPACERANGER_MKGTF") {
+                script "../../mkgtf/main.nf"
+                process {
+                    """
+                    input[0] = file(params.modules_testdata_base_path + 'genomics/homo_sapiens/genome/genome.gtf', checkIfExists: true)
+                    """
+                }
+            }
+        }
+
+        setup {
+            run("SPACERANGER_MKREF") {
+                script "../../mkref/main.nf"
+                process {
+                    """
+                    input[0] = file(params.modules_testdata_base_path + 'genomics/homo_sapiens/genome/genome.fasta', checkIfExists: true)
+                    input[1] = file(params.modules_testdata_base_path + 'genomics/homo_sapiens/genome/genome.gtf', checkIfExists: true)
+                    input[2] = 'homo_sapiens_chr22_reference'
+                    """
+                }
+            }
+        }
+
+        when {
+            process {
+                """
+                input[0] = [
+                    [
+                        id: 'CytAssist_11mm_FFPE_Human_Glioblastoma_2'
+                    ], // Meta map
+                    [
+                        file(params.modules_testdata_base_path + 'genomics/homo_sapiens/10xgenomics/spaceranger/human-brain-cancer-11-mm-capture-area-ffpe-2-standard_v2_ffpe_cytassist/CytAssist_11mm_FFPE_Human_Glioblastoma_2_S1_L001_R1_001.fastq.gz', checkIfExists: true),
+                        file(params.modules_testdata_base_path + 'genomics/homo_sapiens/10xgenomics/spaceranger/human-brain-cancer-11-mm-capture-area-ffpe-2-standard_v2_ffpe_cytassist/CytAssist_11mm_FFPE_Human_Glioblastoma_2_S1_L001_R2_001.fastq.gz', checkIfExists: true)
+                    ], // Reads
+                    [], // Image
+                    'V52Y10-317', // Slide
+                    'B1', // Area
+                    file(params.modules_testdata_base_path + 'genomics/homo_sapiens/10xgenomics/spaceranger/human-brain-cancer-11-mm-capture-area-ffpe-2-standard_v2_ffpe_cytassist/CytAssist_11mm_FFPE_Human_Glioblastoma_image.tif', checkIfExists: true), // Cytaimage
+                    [], // Darkimage
+                    [], // Colorizedimage
+                    [], // Manual alignment (default: automatic alignment)
+                    file('https://s3.us-west-2.amazonaws.com/10x.spatial-slides/gpr/V52Y10/V52Y10-317.gpr') // Slide specification (default: automatic download)
+                ]
+                input[1] = SPACERANGER_MKREF.out.reference // Reference
+                input[2] = file(params.modules_testdata_base_path + 'genomics/homo_sapiens/10xgenomics/spaceranger/human-brain-cancer-11-mm-capture-area-ffpe-2-standard_v2_ffpe_cytassist/CytAssist_11mm_FFPE_Human_Glioblastoma_probe_set.csv', checkIfExists: true) // Probeset (default: use the one included with Space Ranger)
+                """
+            }
+        }
+
+        then {
+            assertAll(
+                { assert process.success },
+                { assert snapshot(
+                    process.out.versions,
+                    process.out.outs.get(0).get(1).findAll { file(it).name !in [
+                        'web_summary.html',
+                        'scalefactors_json.json',
+                        'molecule_info.h5',
+                        'barcodes.tsv.gz',
+                        'features.tsv.gz',
+                        'matrix.mtx.gz',
+                        'cloupe.cloupe'
+                        ]}
+                    ).match()
+                },
+                { assert file(process.out.outs.get(0).get(1).find { file(it).name == 'web_summary.html' }).exists() },
+                { assert file(process.out.outs.get(0).get(1).find { file(it).name == 'scalefactors_json.json' }).exists() },
+                { assert file(process.out.outs.get(0).get(1).find { file(it).name == 'molecule_info.h5' }).exists() },
+                { assert file(process.out.outs.get(0).get(1).find { file(it).name == 'barcodes.tsv.gz' }).exists() },
+                { assert file(process.out.outs.get(0).get(1).find { file(it).name == 'features.tsv.gz' }).exists() },
+                { assert file(process.out.outs.get(0).get(1).find { file(it).name == 'matrix.mtx.gz' }).exists() }
+            )
+        }
+    }
+}
\ No newline at end of file
diff --git a/modules/nf-core/spaceranger/count/tests/main.nf.test.snap b/modules/nf-core/spaceranger/count/tests/main.nf.test.snap
new file mode 100644
index 0000000..dbfaadf
--- /dev/null
+++ b/modules/nf-core/spaceranger/count/tests/main.nf.test.snap
@@ -0,0 +1,90 @@
+{
+    "spaceranger v1 (stub) - homo_sapiens - fasta - gtf - fastq - tif - csv": {
+        "content": [
+            [
+                "versions.yml:md5,4abe169f33d7f99d5d9876b189060aae"
+            ]
+        ],
+        "meta": {
+            "nf-test": "0.9.1",
+            "nextflow": "24.10.4"
+        },
+        "timestamp": "2025-02-13T09:02:47.710975472"
+    },
+    "spaceranger v2 - homo_sapiens - fasta - gtf - fastq - tif - csv": {
+        "content": [
+            [
+                "versions.yml:md5,4abe169f33d7f99d5d9876b189060aae"
+            ],
+            [
+                "clusters.csv:md5,221a4554e62ea94b0df8dbf637d2c13c",
+                "clusters.csv:md5,53ee76645943b5562392aac51d2d9f3f",
+                "clusters.csv:md5,b791359469683ad19cdb8d1af3de5705",
+                "clusters.csv:md5,9a4f9148e0e834c1127bf8393ece6330",
+                "clusters.csv:md5,c11bcc64f870469ab2f136d9272a7a6d",
+                "clusters.csv:md5,488846bbb469365e199928c7a440320a",
+                "clusters.csv:md5,5941f7e847d35a4f06d3631e21d2eb9d",
+                "clusters.csv:md5,d244d405c32766339d2b7a3fa8bf8cee",
+                "clusters.csv:md5,981386408cd953548994c31253e787de",
+                "clusters.csv:md5,24c4f13449e5362fcbcd41b9ff413992",
+                "differential_expression.csv:md5,589c1bd4529f092bb1d332e7da561dad",
+                "differential_expression.csv:md5,d9d978b398b33ac9687b44531909e0cd",
+                "differential_expression.csv:md5,4edbc893280f9d03c3de00a503e86f8c",
+                "differential_expression.csv:md5,316181d501c495384016227309856b09",
+                "differential_expression.csv:md5,dae49941396609fb08df13b82fe89151",
+                "differential_expression.csv:md5,4a13ae44c8454dbcb0298eb63df8b8e8",
+                "differential_expression.csv:md5,eeb02c4afe1f49d5502fb024b25b2c38",
+                "differential_expression.csv:md5,9a456828fe5d762e6e07383da5c2791d",
+                "differential_expression.csv:md5,bcbd1504976824e9f4d20a8dd36e2a1f",
+                "differential_expression.csv:md5,3ad93fc4d52950cfede885dc58cd2823",
+                "components.csv:md5,811a32dce6c795e958dc4bc635ee53be",
+                "dispersion.csv:md5,64c2e57ef0ca9a80cce8b952c81b62f5",
+                "features_selected.csv:md5,bd0c0a20b0b0009df796e8a755d028c1",
+                "projection.csv:md5,e530c925a185965514fa82f4da83fa81",
+                "variance.csv:md5,4159711ab5d64e97737fad9d75d945b3",
+                "projection.csv:md5,ce729f7e237df4570ac3e4a79251df24",
+                "projection.csv:md5,fa7bdefa8424b233fe6461129ab76d57",
+                "filtered_feature_bc_matrix.h5:md5,704256e5150522d9cf2e75e7e47221b6",
+                "metrics_summary.csv:md5,5ece84f5f8e08839749b1c8f2bff6701",
+                "probe_set.csv:md5,5bfb8f12319be1b2b6c14142537c3804",
+                "raw_feature_bc_matrix.h5:md5,ac24486662643ea68562c1a51cbbb2bd",
+                "raw_probe_bc_matrix.h5:md5,8ab08437814506f98e3f10107cfc38ac",
+                "aligned_fiducials.jpg:md5,51dcc3a32d3d5ca4704f664c8ede81ef",
+                "cytassist_image.tiff:md5,0fb04a55e5658f4d158d986a334b034d",
+                "detected_tissue_image.jpg:md5,11c9fa90913b5c6e93cecdb8f53d58db",
+                "spatial_enrichment.csv:md5,4379bc4fef891b45ff9264ee8c408bd0",
+                "tissue_hires_image.png:md5,834706fff299024fab48e6366afc9cb9",
+                "tissue_lowres_image.png:md5,8c1fcb378f7f886301f49ffc4f84360a",
+                "tissue_positions.csv:md5,930aeb2b790032337d91dd27cc70f135"
+            ]
+        ],
+        "meta": {
+            "nf-test": "0.9.1",
+            "nextflow": "24.10.4"
+        },
+        "timestamp": "2025-02-12T11:24:51.984883864"
+    },
+    "spaceranger v1 - homo_sapiens - fasta - gtf - fastq - tif - csv": {
+        "content": [
+            [
+                "versions.yml:md5,4abe169f33d7f99d5d9876b189060aae"
+            ],
+            [
+                "filtered_feature_bc_matrix.h5:md5,649ac955bcb372b0b767013071cca72c",
+                "metrics_summary.csv:md5,38774fc5f54873d711b4898a2dd50e72",
+                "molecule_info.h5:md5,88bb948a426041165b2cc5fe8b180c21",
+                "raw_feature_bc_matrix.h5:md5,63324ae38fbf28bcc2114f170e0fde5d",
+                "aligned_fiducials.jpg:md5,f6217ddd707bb189e665f56b130c3da8",
+                "detected_tissue_image.jpg:md5,c1c7e8741701a576c1ec103c1aaf98ea",
+                "tissue_hires_image.png:md5,d91f8f176ae35ab824ede87117ac0889",
+                "tissue_lowres_image.png:md5,475a04208d193191c84d7a3b5d4eb287",
+                "tissue_positions.csv:md5,7f9cb407b3dd69726a12967b979a5624"
+            ]
+        ],
+        "meta": {
+            "nf-test": "0.9.1",
+            "nextflow": "24.10.4"
+        },
+        "timestamp": "2025-02-12T11:08:30.165324139"
+    }
+}
\ No newline at end of file
diff --git a/modules/nf-core/spaceranger/count/tests/nextflow.config b/modules/nf-core/spaceranger/count/tests/nextflow.config
new file mode 100644
index 0000000..fe9d61a
--- /dev/null
+++ b/modules/nf-core/spaceranger/count/tests/nextflow.config
@@ -0,0 +1,5 @@
+process {
+    withName: SPACERANGER_COUNT {
+        ext.args = '--create-bam false'
+    }
+}
diff --git a/modules/nf-core/spaceranger/mkgtf/main.nf b/modules/nf-core/spaceranger/mkgtf/main.nf
new file mode 100644
index 0000000..86e05cc
--- /dev/null
+++ b/modules/nf-core/spaceranger/mkgtf/main.nf
@@ -0,0 +1,51 @@
+process SPACERANGER_MKGTF {
+    tag "$gtf"
+    label 'process_low'
+
+    container "nf-core/spaceranger:3.1.3"
+
+    input:
+    path gtf
+
+    output:
+    path "*.gtf"         , emit: gtf
+    path "versions.yml"  , emit: versions
+
+    when:
+    task.ext.when == null || task.ext.when
+
+    script:
+    // Exit if running this module with -profile conda / -profile mamba
+    if (workflow.profile.tokenize(',').intersect(['conda', 'mamba']).size() >= 1) {
+        error "SPACERANGER_MKGTF module does not support Conda. Please use Docker / Singularity / Podman instead."
+    }
+    def args = task.ext.args ?: ''
+    def prefix = task.ext.prefix ?: "${gtf.baseName}.filtered"
+    """
+    spaceranger \\
+        mkgtf \\
+        $gtf \\
+        ${prefix}.gtf \\
+        $args
+
+    cat <<-END_VERSIONS > versions.yml
+    "${task.process}":
+        spaceranger: \$(spaceranger -V | sed -e "s/spaceranger spaceranger-//g")
+    END_VERSIONS
+    """
+
+    stub:
+    // Exit if running this module with -profile conda / -profile mamba
+    if (workflow.profile.tokenize(',').intersect(['conda', 'mamba']).size() >= 1) {
+        error "SPACERANGER_COUNT module does not support Conda. Please use Docker / Singularity / Podman instead."
+    }
+    def prefix = task.ext.prefix ?: "${gtf.baseName}.filtered"
+    """
+    touch ${prefix}.gtf
+
+    cat <<-END_VERSIONS > versions.yml
+    "${task.process}":
+        spaceranger: \$(spaceranger -V | sed -e "s/spaceranger spaceranger-//g")
+    END_VERSIONS
+    """
+}
diff --git a/modules/nf-core/spaceranger/mkgtf/meta.yml b/modules/nf-core/spaceranger/mkgtf/meta.yml
new file mode 100644
index 0000000..e68c4af
--- /dev/null
+++ b/modules/nf-core/spaceranger/mkgtf/meta.yml
@@ -0,0 +1,47 @@
+# yaml-language-server: $schema=https://raw.githubusercontent.com/nf-core/modules/master/modules/yaml-schema.json
+name: "spaceranger_mkgtf"
+description: Module to build a filtered GTF needed by the 10x Genomics Space Ranger
+  tool. Uses the spaceranger mkgtf command.
+keywords:
+  - reference
+  - mkref
+  - index
+  - spaceranger
+tools:
+  - "spaceranger":
+      description: |
+        Visium Spatial Gene Expression is a next-generation molecular profiling solution for classifying tissue
+        based on total mRNA. Space Ranger is a set of analysis pipelines that process Visium Spatial Gene Expression
+        data with brightfield and fluorescence microscope images. Space Ranger allows users to map the whole
+        transcriptome in formalin fixed paraffin embedded (FFPE) and fresh frozen tissues to discover novel
+        insights into normal development, disease pathology, and clinical translational research. Space Ranger provides
+        pipelines for end to end analysis of Visium Spatial Gene Expression experiments.
+      homepage: "https://support.10xgenomics.com/spatial-gene-expression/software/pipelines/latest/what-is-space-ranger"
+      documentation: "https://support.10xgenomics.com/spatial-gene-expression/software/pipelines/latest/what-is-space-ranger"
+      tool_dev_url: "https://support.10xgenomics.com/spatial-gene-expression/software/pipelines/latest/what-is-space-ranger"
+      licence:
+        - "10x Genomics EULA"
+      identifier: ""
+input:
+  - gtf:
+      type: file
+      description: The reference GTF transcriptome file
+      pattern: "*.gtf"
+      ontologies: []
+output:
+  gtf:
+    - "*.gtf":
+        type: directory
+        description: The filtered GTF transcriptome file
+        pattern: "*.filtered.gtf"
+  versions:
+    - versions.yml:
+        type: file
+        description: File containing software version
+        pattern: "versions.yml"
+        ontologies:
+          - edam: http://edamontology.org/format_3750 # YAML
+authors:
+  - "@grst"
+maintainers:
+  - "@grst"
diff --git a/modules/nf-core/spaceranger/mkgtf/tests/main.nf.test b/modules/nf-core/spaceranger/mkgtf/tests/main.nf.test
new file mode 100644
index 0000000..7091e0a
--- /dev/null
+++ b/modules/nf-core/spaceranger/mkgtf/tests/main.nf.test
@@ -0,0 +1,59 @@
+nextflow_process {
+
+    name "Test Process SPACERANGER_MKGTF"
+    script "../main.nf"
+    process "SPACERANGER_MKGTF"
+
+    tag "modules"
+    tag "modules_nfcore"
+    tag "spaceranger"
+    tag "spaceranger/mkgtf"
+
+    test("homo_sapiens - gtf") {
+
+        when {
+            process {
+                """
+                input[0] = [
+                    file(params.modules_testdata_base_path + 'genomics/homo_sapiens/genome/genome.gtf', checkIfExists: true)
+                ]
+                """
+            }
+        }
+
+        then {
+            assertAll(
+                { assert process.success },
+                { assert snapshot(
+                    process.out.gtf,
+                    process.out.versions,
+                ).match() }
+            )
+        }
+
+    }
+
+    test("homo_sapiens (stub) - gtf") {
+
+        options "-stub"
+
+        when {
+            process {
+                """
+                input[0] = [
+                    file(params.modules_testdata_base_path + 'genomics/homo_sapiens/genome/genome.gtf', checkIfExists: true)
+                ]
+                """
+            }
+        }
+
+        then {
+            assertAll(
+                { assert process.success },
+                { assert snapshot(process.out.versions).match() },
+            )
+        }
+
+    }
+
+}
\ No newline at end of file
diff --git a/modules/nf-core/spaceranger/mkgtf/tests/main.nf.test.snap b/modules/nf-core/spaceranger/mkgtf/tests/main.nf.test.snap
new file mode 100644
index 0000000..138a739
--- /dev/null
+++ b/modules/nf-core/spaceranger/mkgtf/tests/main.nf.test.snap
@@ -0,0 +1,29 @@
+{
+    "homo_sapiens - gtf": {
+        "content": [
+            [
+                "genome.filtered.gtf:md5,50fc877b1c53b36b3b413aff88bda48c"
+            ],
+            [
+                "versions.yml:md5,2f6c2b1181d43aea5c3fd8095cb181b6"
+            ]
+        ],
+        "meta": {
+            "nf-test": "0.9.1",
+            "nextflow": "24.10.4"
+        },
+        "timestamp": "2025-02-12T16:06:15.384050292"
+    },
+    "homo_sapiens (stub) - gtf": {
+        "content": [
+            [
+                "versions.yml:md5,2f6c2b1181d43aea5c3fd8095cb181b6"
+            ]
+        ],
+        "meta": {
+            "nf-test": "0.9.1",
+            "nextflow": "24.10.4"
+        },
+        "timestamp": "2025-02-12T16:43:38.384121778"
+    }
+}
\ No newline at end of file
diff --git a/subworkflows/local/prepare_ref/main.nf b/subworkflows/local/prepare_ref/main.nf
index 6660156..2d4dc1b 100644
--- a/subworkflows/local/prepare_ref/main.nf
+++ b/subworkflows/local/prepare_ref/main.nf
@@ -5,8 +5,9 @@
 include { GUNZIP as GUNZIP_FASTA            } from '../../../modules/nf-core/gunzip'
 include { GUNZIP as GUNZIP_GTF              } from '../../../modules/nf-core/gunzip'
 include { GUNZIP as GUNZIP_GFF              } from '../../../modules/nf-core/gunzip'
-include { UNTAR as UNTAR_STAR_INDEX         } from '../../../modules/nf-core/untar'
+include { UNTAR as UNTAR_SPACERANGER_REF    } from "../../../modules/nf-core/untar"
 include { GFFREAD                           } from '../../../modules/nf-core/gffread'
+include { SPACERANGER_MKGTF                 } from '../../../modules/nf-core/spaceranger/mkgtf'
 include { SPACERANGER_MKREF                 } from '../../../modules/nf-core/spaceranger/mkref'
 
 workflow PREPARE_REF {
@@ -28,8 +29,21 @@ workflow PREPARE_REF {
 
     if (params.spaceranger_index) {
 
+        ch_fasta = Channel.empty()
+        ch_gtf   = Channel.empty()
+
         // Define spaceranger index channel from the user-provided one
-        ch_spaceranger_index = spaceranger_index
+        if (params.spaceranger_index ==~ /.*\.tar\.gz$/) {
+            ref_file = file(params.spaceranger_index)
+            UNTAR_SPACERANGER_REF ([
+                ["id": file(params.spaceranger_index).name.replaceAll(/\.(tar)(\.gz)?$/, '')],
+                ref_file
+            ])
+            ch_spaceranger_index = UNTAR_SPACERANGER_REF.out.untar.map{ it[1] }
+            ch_versions = ch_versions.mix(UNTAR_SPACERANGER_REF.out.versions)
+        } else {
+            ch_spaceranger_index = file(params.spaceranger_index, type: "dir", checkIfExists: true)
+        }
 
     } else {
 
@@ -65,12 +79,20 @@ workflow PREPARE_REF {
             ch_fasta = fasta
         }
 
+        //
+        // Prepare gft file by keeping specific biotypes
+        //
+        SPACERANGER_MKGTF(
+            ch_gtf,
+        )
+        ch_gtf_filtered = SPACERANGER_MKGTF.out.gtf
+
         //
         // Create Spacer Ranger reference
         //
         SPACERANGER_MKREF(
             ch_fasta,
-            ch_gtf,
+            ch_gtf_filtered,
             file(params.fasta).name.replaceAll(/\.(fa|fasta)(\.gz)?$/, '')
         )
 
diff --git a/workflows/spatialomics.nf b/workflows/spatialomics.nf
index a6763a6..ad5ebf9 100644
--- a/workflows/spatialomics.nf
+++ b/workflows/spatialomics.nf
@@ -5,12 +5,15 @@
 */
 include { FASTQC                 } from '../modules/nf-core/fastqc/main'
 include { MULTIQC                } from '../modules/nf-core/multiqc/main'
+include { SPACERANGER_COUNT      } from '../modules/nf-core/spaceranger/count/main'
+
+include { PREPARE_REF            } from '../subworkflows/local/prepare_ref'
+include { PREPARE_FASTQ          } from '../subworkflows/local/prepare_fastq'
+
 include { paramsSummaryMap       } from 'plugin/nf-schema'
 include { paramsSummaryMultiqc   } from '../subworkflows/nf-core/utils_nfcore_pipeline'
 include { softwareVersionsToYAML } from '../subworkflows/nf-core/utils_nfcore_pipeline'
 include { methodsDescriptionText } from '../subworkflows/local/utils_nfcore_spatialomics_pipeline'
-include { PREPARE_REF            } from '../subworkflows/local/prepare_ref'
-include { PREPARE_FASTQ          } from '../subworkflows/local/prepare_fastq'
 
 /*
 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
@@ -26,6 +29,7 @@ workflow SPATIALOMICS {
     ch_gtf                // value channel: path(gtf)
     ch_gff                // value channel: path(gff)
     ch_spaceranger_index  // value channel: path(spaceranger_index)
+    ch_probeset           // value channel: path(probeset) (optional)
 
     main:
 
@@ -53,6 +57,27 @@ workflow SPATIALOMICS {
     ch_versions = ch_versions.mix(PREPARE_FASTQ.out.versions)
     ch_multiqc_files = ch_multiqc_files.mix(PREPARE_FASTQ.out.multiqc_files)
 
+    ch_reads
+        .multiMap { meta, fastq ->
+            reads: [ ["id": meta.id], fastq ]
+            slide_and_img: [ ["id": meta.id], meta.image, meta.slide, meta.area, meta.cytaimage, meta.darkimage, meta.colorizedimage, meta.alignment, meta.slidefile ]
+        }
+        .set { ch_reads_with_meta }
+
+    ch_reads_with_meta.reads.view()
+    ch_reads_with_meta.slide_and_img.view()
+
+    //
+    // MODULE: Align and quantify with Space Ranger
+    //
+    SPACERANGER_COUNT(
+        ch_reads_with_meta.reads,
+        ch_reads_with_meta.slide_and_img,
+        PREPARE_REF.out.spaceranger_index,
+        ch_probeset
+    )
+    ch_versions = ch_versions.mix(SPACERANGER_COUNT.out.versions)
+
     //
     // Collate and save software versions
     //

From 0c4a02191bd3651961d4e712b57ac908f6763850 Mon Sep 17 00:00:00 2001
From: Matteo Bonfanti <matteo.bonfanti@fht.org>
Date: Tue, 21 Oct 2025 16:35:08 +0200
Subject: [PATCH 02/18] adding index name as parameter

---
 conf/modules.config                    |  2 +-
 subworkflows/local/prepare_ref/main.nf | 11 +++++++----
 workflows/spatialomics.nf              |  1 +
 3 files changed, 9 insertions(+), 5 deletions(-)

diff --git a/conf/modules.config b/conf/modules.config
index 97c2546..3df6b91 100644
--- a/conf/modules.config
+++ b/conf/modules.config
@@ -61,7 +61,7 @@ process {
             mode: params.publish_dir_mode,
             saveAs: { filename -> filename.equals('versions.yml') ? null : filename }
         ]
-        ext.args   = "--create-bam false"
+        ext.args   = "--create-bam true"
         time   = { 240.h * task.attempt }
     }
 
diff --git a/subworkflows/local/prepare_ref/main.nf b/subworkflows/local/prepare_ref/main.nf
index 2d4dc1b..a8f5cb4 100644
--- a/subworkflows/local/prepare_ref/main.nf
+++ b/subworkflows/local/prepare_ref/main.nf
@@ -17,6 +17,7 @@ workflow PREPARE_REF {
     gtf                      // file: /path/to/genome.gtf
     gff                      // file: /path/to/genome.gff
     spaceranger_index        // directory: /path/to/spaceranger/index/ (optional!)
+    reference_name           // string: name for the new reference (if building new index)
 
     main:
 
@@ -24,6 +25,9 @@ workflow PREPARE_REF {
         "Must provide a the spaceranger index (--spaceranger_index) \
         or a fasta file ('--fasta') and a gtf/gff file ('--gtf'/'--gff') if no index is given!"
 
+    assert (params.spaceranger_index) || (reference_name):
+        "Must provide a reference name (--reference_name) when building a new spaceranger index!"
+
     // Versions collector
     ch_versions = Channel.empty()
 
@@ -34,15 +38,14 @@ workflow PREPARE_REF {
 
         // Define spaceranger index channel from the user-provided one
         if (params.spaceranger_index ==~ /.*\.tar\.gz$/) {
-            ref_file = file(params.spaceranger_index)
             UNTAR_SPACERANGER_REF ([
                 ["id": file(params.spaceranger_index).name.replaceAll(/\.(tar)(\.gz)?$/, '')],
-                ref_file
+                spaceranger_index
             ])
             ch_spaceranger_index = UNTAR_SPACERANGER_REF.out.untar.map{ it[1] }
             ch_versions = ch_versions.mix(UNTAR_SPACERANGER_REF.out.versions)
         } else {
-            ch_spaceranger_index = file(params.spaceranger_index, type: "dir", checkIfExists: true)
+            ch_spaceranger_index = spaceranger_index
         }
 
     } else {
@@ -93,7 +96,7 @@ workflow PREPARE_REF {
         SPACERANGER_MKREF(
             ch_fasta,
             ch_gtf_filtered,
-            file(params.fasta).name.replaceAll(/\.(fa|fasta)(\.gz)?$/, '')
+            reference_name
         )
 
         // Channel to handle SPACERANGER_MKREF output
diff --git a/workflows/spatialomics.nf b/workflows/spatialomics.nf
index ad5ebf9..3e62a61 100644
--- a/workflows/spatialomics.nf
+++ b/workflows/spatialomics.nf
@@ -44,6 +44,7 @@ workflow SPATIALOMICS {
         ch_gtf,
         ch_gff,
         ch_spaceranger_index,
+        params.reference_name
     )
     ch_versions = ch_versions.mix(PREPARE_REF.out.versions)
 

From 25e6d30e15d8fc08b7dd7813fd1302a44a6c659d Mon Sep 17 00:00:00 2001
From: Matteo Bonfanti <matteo.bonfanti@fht.org>
Date: Tue, 21 Oct 2025 16:39:57 +0200
Subject: [PATCH 03/18] removing view()

---
 workflows/spatialomics.nf | 3 ---
 1 file changed, 3 deletions(-)

diff --git a/workflows/spatialomics.nf b/workflows/spatialomics.nf
index 3e62a61..4d848ce 100644
--- a/workflows/spatialomics.nf
+++ b/workflows/spatialomics.nf
@@ -65,9 +65,6 @@ workflow SPATIALOMICS {
         }
         .set { ch_reads_with_meta }
 
-    ch_reads_with_meta.reads.view()
-    ch_reads_with_meta.slide_and_img.view()
-
     //
     // MODULE: Align and quantify with Space Ranger
     //

From a6d5c7c13c08db3064208a9e4fd67a05ad43e78e Mon Sep 17 00:00:00 2001
From: Matteo Bonfanti <matteo.bonfanti@fht.org>
Date: Mon, 3 Nov 2025 14:46:09 +0100
Subject: [PATCH 04/18] new test for visium

---
 assets/samplesheet_test.csv | 3 +--
 conf/test.config            | 8 +++++---
 2 files changed, 6 insertions(+), 5 deletions(-)

diff --git a/assets/samplesheet_test.csv b/assets/samplesheet_test.csv
index 4aa0b09..6e433f0 100644
--- a/assets/samplesheet_test.csv
+++ b/assets/samplesheet_test.csv
@@ -1,3 +1,2 @@
 sample,fastq_1,fastq_2,image,cytaimage,slide,area,darkimage,colorizedimage,manual_alignment,slidefile
-Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2,https://raw.githubusercontent.com/nf-core/test-datasets/spatialvi/testdata/human-lung-cancer-post-xenium_hd_ffpe/Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2_S1_L001_R1_001.fastq.gz,https://raw.githubusercontent.com/nf-core/test-datasets/spatialvi/testdata/human-lung-cancer-post-xenium_hd_ffpe/Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2_S1_L001_R2_001.fastq.gz,https://raw.githubusercontent.com/nf-core/test-datasets/spatialvi/testdata/human-lung-cancer-post-xenium_hd_ffpe/Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2_tissue_image.btf,https://raw.githubusercontent.com/nf-core/test-datasets/spatialvi/testdata/human-lung-cancer-post-xenium_hd_ffpe/Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2_image.tif,H1-84QJZFR,D1,,,,,
-Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2,https://raw.githubusercontent.com/nf-core/test-datasets/spatialvi/testdata/human-lung-cancer-post-xenium_hd_ffpe/Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2_S1_L002_R1_001.fastq.gz,https://raw.githubusercontent.com/nf-core/test-datasets/spatialvi/testdata/human-lung-cancer-post-xenium_hd_ffpe/Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2_S1_L002_R2_001.fastq.gz,https://raw.githubusercontent.com/nf-core/test-datasets/spatialvi/testdata/human-lung-cancer-post-xenium_hd_ffpe/Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2_tissue_image.btf,https://raw.githubusercontent.com/nf-core/test-datasets/spatialvi/testdata/human-lung-cancer-post-xenium_hd_ffpe/Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2_image.tif,H1-84QJZFR,D1,,,,,
+Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2,https://raw.githubusercontent.com/nfdata-omics/test-datasets/spatialomics/Visium_HD_Human_Colon_Cancer/Visium_HD_Human_Colon_Cancer_chr21_sub40000_P2_S2_L001_R1_001.fastq.gz,https://raw.githubusercontent.com/nfdata-omics/test-datasets/spatialomics/Visium_HD_Human_Colon_Cancer/Visium_HD_Human_Colon_Cancer_chr21_sub40000_P2_S2_L001_R2_001.fastq.gz,,https://raw.githubusercontent.com/nfdata-omics/test-datasets/spatialomics/Visium_HD_Human_Colon_Cancer/Visium_HD_Human_Colon_Cancer_P2_image.tif,H1-VM2JXXK,A1,,,,
diff --git a/conf/test.config b/conf/test.config
index 82c4d57..86a2b82 100644
--- a/conf/test.config
+++ b/conf/test.config
@@ -24,8 +24,10 @@ params {
 
     // Input and output
     input             = "${projectDir}/assets/samplesheet_test.csv"
-    probeset          = "https://raw.githubusercontent.com/nf-core/test-datasets/spatialvi/testdata/human-lung-cancer-post-xenium_hd_ffpe/Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2_probe_set.csv"
-    spaceranger_index = "https://raw.githubusercontent.com/nf-core/test-datasets/spatialvi/testdata/GRCh38.tar.gz"
-
+    probeset          = "https://raw.githubusercontent.com/nfdata-omics/test-datasets/spatialomics/Visium_HD_Human_Colon_Cancer/GSM8594568_P2CRC_probe_set.csv"
+    fasta             = "https://raw.githubusercontent.com/nfdata-omics/test-datasets/refs/chr21-cellranger/chr21.fa.gz"
+    gtf               = "https://raw.githubusercontent.com/nfdata-omics/test-datasets/refs/chr21-cellranger/chr21.gtf.gz"
+    reference_name    = "GRCh38"
 
 }
+

From d7d8366f511cbbb960ee6d1c69c86a447b5a59d6 Mon Sep 17 00:00:00 2001
From: Matteo Bonfanti <matteo.bonfanti@fht.org>
Date: Mon, 3 Nov 2025 15:10:08 +0100
Subject: [PATCH 05/18] fixed linting and parameter validation

---
 main.nf                                       |  2 +-
 modules.json                                  |  3 +-
 .../spaceranger/count/spaceranger-count.diff  | 29 +++++++++++++++++++
 nextflow_schema.json                          | 21 +++++++++++++-
 subworkflows/local/prepare_ref/main.nf        |  2 ++
 5 files changed, 54 insertions(+), 3 deletions(-)
 create mode 100644 modules/nf-core/spaceranger/count/spaceranger-count.diff

diff --git a/main.nf b/main.nf
index 0624405..d19c6b9 100644
--- a/main.nf
+++ b/main.nf
@@ -25,7 +25,7 @@ include { getGenomeAttribute      } from './subworkflows/local/utils_nfcore_spat
 */
 
 params.fasta             = getGenomeAttribute('fasta')
-params.gft               = getGenomeAttribute('gtf')
+params.gtf               = getGenomeAttribute('gtf')
 params.spaceranger_index = getGenomeAttribute('spaceranger_index')
 
 /*
diff --git a/modules.json b/modules.json
index dbc41c1..1f1da73 100644
--- a/modules.json
+++ b/modules.json
@@ -38,7 +38,8 @@
                     "spaceranger/count": {
                         "branch": "master",
                         "git_sha": "41dfa3f7c0ffabb96a6a813fe321c6d1cc5b6e46",
-                        "installed_by": ["modules"]
+                        "installed_by": ["modules"],
+                        "patch": "modules/nf-core/spaceranger/count/spaceranger-count.diff"
                     },
                     "spaceranger/mkgtf": {
                         "branch": "master",
diff --git a/modules/nf-core/spaceranger/count/spaceranger-count.diff b/modules/nf-core/spaceranger/count/spaceranger-count.diff
new file mode 100644
index 0000000..9829a15
--- /dev/null
+++ b/modules/nf-core/spaceranger/count/spaceranger-count.diff
@@ -0,0 +1,29 @@
+Changes in component 'nf-core/spaceranger/count'
+'modules/nf-core/spaceranger/count/meta.yml' is unchanged
+Changes in 'spaceranger/count/main.nf':
+--- modules/nf-core/spaceranger/count/main.nf
++++ modules/nf-core/spaceranger/count/main.nf
+@@ -5,7 +5,8 @@
+     container "nf-core/spaceranger:3.1.3"
+ 
+     input:
+-    tuple val(meta), path(reads), path(image), val(slide), val(area), path(cytaimage), path(darkimage), path(colorizedimage), path(alignment), path(slidefile)
++    tuple val(meta), path("fastqs/${meta.id}_S1_L001_R?_001.fastq.gz")
++    tuple val(meta2), path(image), val(slide), val(area), path(cytaimage), path(darkimage), path(colorizedimage), path(alignment), path(slidefile)
+     path(reference)
+     path(probeset)
+ 
+@@ -40,7 +41,7 @@
+     spaceranger count \\
+         --id="${prefix}" \\
+         --sample="${meta.id}" \\
+-        --fastqs=. \\
++        --fastqs=fastqs \\
+         --transcriptome="${reference}" \\
+         --localcores=${task.cpus} \\
+         --localmem=${task.memory.toGiga()} \\
+
+'modules/nf-core/spaceranger/count/tests/nextflow.config' is unchanged
+'modules/nf-core/spaceranger/count/tests/main.nf.test.snap' is unchanged
+'modules/nf-core/spaceranger/count/tests/main.nf.test' is unchanged
+************************************************************
diff --git a/nextflow_schema.json b/nextflow_schema.json
index b919498..a3c2528 100644
--- a/nextflow_schema.json
+++ b/nextflow_schema.json
@@ -62,9 +62,28 @@
                     "mimetype": "text/plain",
                     "pattern": "^\\S+\\.fn?a(sta)?(\\.gz)?$",
                     "description": "Path to FASTA genome file.",
-                    "help_text": "This parameter is *mandatory* if `--genome` is not specified. If you don't have a BWA index available this will be generated for you automatically. Combine with `--save_reference` to save BWA index for future runs.",
+                    "help_text": "This parameter is *mandatory* if `--genome` or `--spaceranger_index` are not specified. If you don't have a Space Ranger index available this will be generated for you automatically.",
                     "fa_icon": "far fa-file-code"
                 },
+                "gtf": {
+                    "type": "string",
+                    "format": "file-path",
+                    "exists": true,
+                    "mimetype": "text/plain",
+                    "pattern": "^\\S+\\.gtf(\\.gz)?$",
+                    "description": "Path to GTF annotation file.",
+                    "help_text": "This parameter is mandatory if `--genome` or `--spaceranger_index` are not specified. If you don't have a Space Ranger index available this will be generated for you automatically.",
+                    "errorMessage": "The GTF file must have a .gtf or .gtf.gz extension, must not contain spaces, and must exist.",
+                    "fa_icon": "fas fa-code-branch"
+                },
+                "spaceranger_index": {
+                    "type": "string",
+                    "format": "path",
+                    "description": "Path to a Space Ranger reference folder. Can alternatively be provided as `tar.gz` archive.",
+                    "help_text": "Please see the [10x website](https://support.10xgenomics.com/spatial-gene-expression/software/downloads/latest) to download either of the supported human or mouse references.",
+                    "fa_icon": "fas fa-folder-open",
+                    "exists": true
+                },
                 "igenomes_ignore": {
                     "type": "boolean",
                     "description": "Do not load the iGenomes reference config.",
diff --git a/subworkflows/local/prepare_ref/main.nf b/subworkflows/local/prepare_ref/main.nf
index a8f5cb4..fc0daad 100644
--- a/subworkflows/local/prepare_ref/main.nf
+++ b/subworkflows/local/prepare_ref/main.nf
@@ -88,6 +88,7 @@ workflow PREPARE_REF {
         SPACERANGER_MKGTF(
             ch_gtf,
         )
+        ch_versions = ch_versions.mix(SPACERANGER_MKGTF.out.versions)
         ch_gtf_filtered = SPACERANGER_MKGTF.out.gtf
 
         //
@@ -98,6 +99,7 @@ workflow PREPARE_REF {
             ch_gtf_filtered,
             reference_name
         )
+        ch_versions = ch_versions.mix(SPACERANGER_MKREF.out.versions)
 
         // Channel to handle SPACERANGER_MKREF output
         ch_spaceranger_index = SPACERANGER_MKREF.out.reference.ifEmpty {

From 4643cd49e1ded95cf03865b6d714d89ca7fe85e0 Mon Sep 17 00:00:00 2001
From: Matteo Bonfanti <matteo.bonfanti@fht.org>
Date: Mon, 3 Nov 2025 15:46:37 +0100
Subject: [PATCH 06/18] updated test snap and validation schema

---
 nextflow.config            |   2 +
 nextflow_schema.json       |  14 ++
 tests/default.nf.test.snap | 427 ++++++++++++++++++++++++++++++++-----
 3 files changed, 389 insertions(+), 54 deletions(-)

diff --git a/nextflow.config b/nextflow.config
index 44b6c82..b21db1b 100644
--- a/nextflow.config
+++ b/nextflow.config
@@ -17,6 +17,8 @@ params {
     genome                     = null
     igenomes_base              = 's3://ngi-igenomes/igenomes/'
     igenomes_ignore            = false
+    reference_name             = null
+    probeset                   = null
 
     // MultiQC options
     multiqc_config             = null
diff --git a/nextflow_schema.json b/nextflow_schema.json
index a3c2528..1efea2f 100644
--- a/nextflow_schema.json
+++ b/nextflow_schema.json
@@ -84,6 +84,20 @@
                     "fa_icon": "fas fa-folder-open",
                     "exists": true
                 },
+                "reference_name": {
+                    "type": "string",
+                    "fa_icon": "fas fa-book",
+                    "description": "Genome assembly label that will be used to name a new index built from fasta and gft files with spaceranger mkref."
+                },
+                "probeset": {
+                    "type": "string",
+                    "format": "file-path",
+                    "mimetype": "text/csv",
+                    "pattern": "^\\S+\\.csv$",
+                    "description": "Location of Space Ranger probeset file.",
+                    "fa_icon": "fas fa-file-csv",
+                    "exists": true
+                },
                 "igenomes_ignore": {
                     "type": "boolean",
                     "description": "Do not load the iGenomes reference config.",
diff --git a/tests/default.nf.test.snap b/tests/default.nf.test.snap
index 0eab4d0..eb1a0b7 100644
--- a/tests/default.nf.test.snap
+++ b/tests/default.nf.test.snap
@@ -11,11 +11,209 @@
                 "FQ_LINT": {
                     "fq": "0.12.0 (2024-07-08)"
                 },
+                "GUNZIP_FASTA": {
+                    "gunzip": 1.13
+                },
+                "GUNZIP_GTF": {
+                    "gunzip": 1.13
+                },
+                "SPACERANGER_COUNT": {
+                    "spaceranger": "3.1.3"
+                },
+                "SPACERANGER_MKGTF": {
+                    "spaceranger": "3.1.3"
+                },
+                "SPACERANGER_MKREF": {
+                    "spaceranger": "3.1.3"
+                },
                 "Workflow": {
                     "nfdata-omics/spatialomics": "v0.0.1dev"
                 }
             },
             [
+                "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2",
+                "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/count",
+                "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/count/outs",
+                "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/count/outs/binned_outputs",
+                "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/count/outs/binned_outputs/square_002um",
+                "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/count/outs/binned_outputs/square_002um/filtered_feature_bc_matrix",
+                "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/count/outs/binned_outputs/square_002um/filtered_feature_bc_matrix.h5",
+                "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/count/outs/binned_outputs/square_002um/filtered_feature_bc_matrix/barcodes.tsv.gz",
+                "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/count/outs/binned_outputs/square_002um/filtered_feature_bc_matrix/features.tsv.gz",
+                "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/count/outs/binned_outputs/square_002um/filtered_feature_bc_matrix/matrix.mtx.gz",
+                "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/count/outs/binned_outputs/square_002um/raw_feature_bc_matrix",
+                "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/count/outs/binned_outputs/square_002um/raw_feature_bc_matrix.h5",
+                "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/count/outs/binned_outputs/square_002um/raw_feature_bc_matrix/barcodes.tsv.gz",
+                "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/count/outs/binned_outputs/square_002um/raw_feature_bc_matrix/features.tsv.gz",
+                "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/count/outs/binned_outputs/square_002um/raw_feature_bc_matrix/matrix.mtx.gz",
+                "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/count/outs/binned_outputs/square_002um/raw_probe_bc_matrix.h5",
+                "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/count/outs/binned_outputs/square_002um/spatial",
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+                "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/count/outs/binned_outputs/square_016um/raw_feature_bc_matrix/matrix.mtx.gz",
+                "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/count/outs/binned_outputs/square_016um/spatial",
+                "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/count/outs/binned_outputs/square_016um/spatial/aligned_fiducials.jpg",
+                "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/count/outs/binned_outputs/square_016um/spatial/cytassist_image.tiff",
+                "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/count/outs/binned_outputs/square_016um/spatial/detected_tissue_image.jpg",
+                "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/count/outs/binned_outputs/square_016um/spatial/scalefactors_json.json",
+                "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/count/outs/binned_outputs/square_016um/spatial/tissue_hires_image.png",
+                "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/count/outs/binned_outputs/square_016um/spatial/tissue_lowres_image.png",
+                "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/count/outs/binned_outputs/square_016um/spatial/tissue_positions.parquet",
+                "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/count/outs/cloupe_008um.cloupe",
+                "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/count/outs/feature_slice.h5",
+                "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/count/outs/metrics_summary.csv",
+                "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/count/outs/molecule_info.h5",
+                "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/count/outs/possorted_genome_bam.bam",
+                "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/count/outs/possorted_genome_bam.bam.bai",
+                "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/count/outs/probe_set.csv",
+                "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/count/outs/spatial",
+                "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/count/outs/spatial/aligned_fiducials.jpg",
+                "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/count/outs/spatial/cytassist_image.tiff",
+                "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/count/outs/spatial/detected_tissue_image.jpg",
+                "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/count/outs/spatial/tissue_hires_image.png",
+                "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/count/outs/spatial/tissue_lowres_image.png",
+                "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/count/outs/web_summary.html",
                 "cat",
                 "cat/Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2_1.merged.fastq.gz",
                 "cat/Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2_2.merged.fastq.gz",
@@ -26,6 +224,9 @@
                 "fastqc/Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2_2_fastqc.zip",
                 "fq",
                 "fq/Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2.fq_lint.txt",
+                "gunzip",
+                "gunzip/chr21.fa",
+                "gunzip/chr21.gtf",
                 "multiqc",
                 "multiqc/multiqc_data",
                 "multiqc/multiqc_data/fastqc-status-check-heatmap.txt",
@@ -87,72 +288,190 @@
                 "multiqc/multiqc_report.html",
                 "pipeline_info",
                 "pipeline_info/spatialomics_software_mqc_versions.yml",
-                "spaceranger",
-                "spaceranger/GRCh38_chr21",
-                "spaceranger/GRCh38_chr21/fasta",
-                "spaceranger/GRCh38_chr21/fasta/genome.fa",
-                "spaceranger/GRCh38_chr21/fasta/genome.fa.fai",
-                "spaceranger/GRCh38_chr21/genes",
-                "spaceranger/GRCh38_chr21/genes/genes.gtf.gz",
-                "spaceranger/GRCh38_chr21/reference.json",
-                "spaceranger/GRCh38_chr21/star",
-                "spaceranger/GRCh38_chr21/star/Genome",
-                "spaceranger/GRCh38_chr21/star/SA",
-                "spaceranger/GRCh38_chr21/star/SAindex",
-                "spaceranger/GRCh38_chr21/star/chrLength.txt",
-                "spaceranger/GRCh38_chr21/star/chrName.txt",
-                "spaceranger/GRCh38_chr21/star/chrNameLength.txt",
-                "spaceranger/GRCh38_chr21/star/chrStart.txt",
-                "spaceranger/GRCh38_chr21/star/exonGeTrInfo.tab",
-                "spaceranger/GRCh38_chr21/star/exonInfo.tab",
-                "spaceranger/GRCh38_chr21/star/geneInfo.tab",
-                "spaceranger/GRCh38_chr21/star/genomeParameters.txt",
-                "spaceranger/GRCh38_chr21/star/sjdbInfo.txt",
-                "spaceranger/GRCh38_chr21/star/sjdbList.fromGTF.out.tab",
-                "spaceranger/GRCh38_chr21/star/sjdbList.out.tab",
-                "spaceranger/GRCh38_chr21/star/transcriptInfo.tab"
+                "reference",
+                "reference/mkgtf",
+                "reference/mkgtf/chr21.filtered.gtf",
+                "reference/mkref",
+                "reference/mkref/GRCh38",
+                "reference/mkref/GRCh38/fasta",
+                "reference/mkref/GRCh38/fasta/genome.fa",
+                "reference/mkref/GRCh38/fasta/genome.fa.fai",
+                "reference/mkref/GRCh38/genes",
+                "reference/mkref/GRCh38/genes/genes.gtf.gz",
+                "reference/mkref/GRCh38/reference.json",
+                "reference/mkref/GRCh38/star",
+                "reference/mkref/GRCh38/star/Genome",
+                "reference/mkref/GRCh38/star/SA",
+                "reference/mkref/GRCh38/star/SAindex",
+                "reference/mkref/GRCh38/star/chrLength.txt",
+                "reference/mkref/GRCh38/star/chrName.txt",
+                "reference/mkref/GRCh38/star/chrNameLength.txt",
+                "reference/mkref/GRCh38/star/chrStart.txt",
+                "reference/mkref/GRCh38/star/exonGeTrInfo.tab",
+                "reference/mkref/GRCh38/star/exonInfo.tab",
+                "reference/mkref/GRCh38/star/geneInfo.tab",
+                "reference/mkref/GRCh38/star/genomeParameters.txt",
+                "reference/mkref/GRCh38/star/sjdbInfo.txt",
+                "reference/mkref/GRCh38/star/sjdbList.fromGTF.out.tab",
+                "reference/mkref/GRCh38/star/sjdbList.out.tab",
+                "reference/mkref/GRCh38/star/transcriptInfo.tab"
             ],
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+                "genome.fa:md5,a7a8ee17d735d23160137c7793f9f872",
+                "genome.fa.fai:md5,a7e52efeee796ff94ede575403a31586",
+                "genes.gtf.gz:md5,d2db40ac04bf0bce710a304139ca297b",
+                "reference.json:md5,5b5a497cd6bd824b68e305176e9f7625",
+                "Genome:md5,ddad1b8708d0fe4624f0d945f1e53381",
+                "SA:md5,f01f7cd63c62a6b69ad49a9919653a9a",
+                "SAindex:md5,fd7b706c62781e92b759d5e8aa5151a7",
                 "chrLength.txt:md5,b0be0a56ddefa84552742c72d4859eac",
                 "chrName.txt:md5,e99d7d1051eee43ceab5563c2d09fcee",
                 "chrNameLength.txt:md5,c985a141685e8431ec27c782816cb744",
                 "chrStart.txt:md5,6925b594ea2eeb964ba87cd6d42ab98f",
-                "exonGeTrInfo.tab:md5,a36b92eeceaaf921b9d19cd8e806a98e",
-                "exonInfo.tab:md5,bb892190d1ebdd59bf55652916121479",
-                "geneInfo.tab:md5,4303e97d841035b5c0fcc3686b2f5a36",
-                "genomeParameters.txt:md5,1efd18b59cae82e3cc63f9ffc25ff85b",
-                "sjdbInfo.txt:md5,12fb05dc7cea89735a0c19e1c0df61cb",
-                "sjdbList.fromGTF.out.tab:md5,d9e4a184cde15a5ab282ff66e740a4a2",
-                "sjdbList.out.tab:md5,766fbca932681f8666b3a9e5fb3640bd",
-                "transcriptInfo.tab:md5,ad9baa68c5432908b42693edb9aed02a"
+                "exonGeTrInfo.tab:md5,19506a649344a3dbb33bfa47d794851b",
+                "exonInfo.tab:md5,30350358691756b50407c0f8cb6ce5b3",
+                "geneInfo.tab:md5,67cb5583677c9b6f4c5490c6a89dba87",
+                "genomeParameters.txt:md5,195a73612305560d9d2c68c046518727",
+                "sjdbInfo.txt:md5,a544cc53ebf721c5d55d1995abcc907e",
+                "sjdbList.fromGTF.out.tab:md5,3427299e4047e7ba0404e07fb0ac03ee",
+                "sjdbList.out.tab:md5,874227af46e44a89ec5fdbe25d3154a5",
+                "transcriptInfo.tab:md5,a86211e41ef8fd5bcce871ebbbe39638"
             ]
         ],
         "meta": {
             "nf-test": "0.9.2",
             "nextflow": "25.04.8"
         },
-        "timestamp": "2025-10-20T14:57:14.219336507"
+        "timestamp": "2025-11-03T15:37:00.437153276"
     }
 }
\ No newline at end of file

From 88dbd459c12138e80c4810ff065a230b3c02532e Mon Sep 17 00:00:00 2001
From: Matteo Bonfanti <matteo.bonfanti@fht.org>
Date: Mon, 3 Nov 2025 15:50:22 +0100
Subject: [PATCH 07/18] fixing eof line in test.config

---
 conf/test.config | 3 +--
 1 file changed, 1 insertion(+), 2 deletions(-)

diff --git a/conf/test.config b/conf/test.config
index 86a2b82..51cb49a 100644
--- a/conf/test.config
+++ b/conf/test.config
@@ -22,7 +22,7 @@ params {
     config_profile_name        = 'Test profile'
     config_profile_description = 'Minimal test dataset to check pipeline function'
 
-    // Input and output
+    // Input
     input             = "${projectDir}/assets/samplesheet_test.csv"
     probeset          = "https://raw.githubusercontent.com/nfdata-omics/test-datasets/spatialomics/Visium_HD_Human_Colon_Cancer/GSM8594568_P2CRC_probe_set.csv"
     fasta             = "https://raw.githubusercontent.com/nfdata-omics/test-datasets/refs/chr21-cellranger/chr21.fa.gz"
@@ -30,4 +30,3 @@ params {
     reference_name    = "GRCh38"
 
 }
-

From f5327beb650e377e1f30cc7ef40a1332e3753c2a Mon Sep 17 00:00:00 2001
From: Matteo Bonfanti <matteo.bonfanti@fht.org>
Date: Tue, 4 Nov 2025 12:15:18 +0100
Subject: [PATCH 08/18] reorganization of tests

---
 assets/samplesheet_test.csv      |  3 ++-
 assets/samplesheet_test_long.csv |  2 ++
 conf/test.config                 |  6 ++----
 conf/test_long.config            | 32 ++++++++++++++++++++++++++++++++
 nextflow.config                  |  1 +
 5 files changed, 39 insertions(+), 5 deletions(-)
 create mode 100644 assets/samplesheet_test_long.csv
 create mode 100644 conf/test_long.config

diff --git a/assets/samplesheet_test.csv b/assets/samplesheet_test.csv
index 6e433f0..8e0c992 100644
--- a/assets/samplesheet_test.csv
+++ b/assets/samplesheet_test.csv
@@ -1,2 +1,3 @@
 sample,fastq_1,fastq_2,image,cytaimage,slide,area,darkimage,colorizedimage,manual_alignment,slidefile
-Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2,https://raw.githubusercontent.com/nfdata-omics/test-datasets/spatialomics/Visium_HD_Human_Colon_Cancer/Visium_HD_Human_Colon_Cancer_chr21_sub40000_P2_S2_L001_R1_001.fastq.gz,https://raw.githubusercontent.com/nfdata-omics/test-datasets/spatialomics/Visium_HD_Human_Colon_Cancer/Visium_HD_Human_Colon_Cancer_chr21_sub40000_P2_S2_L001_R2_001.fastq.gz,,https://raw.githubusercontent.com/nfdata-omics/test-datasets/spatialomics/Visium_HD_Human_Colon_Cancer/Visium_HD_Human_Colon_Cancer_P2_image.tif,H1-VM2JXXK,A1,,,,
+Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2,https://raw.githubusercontent.com/nf-core/test-datasets/spatialvi/testdata/human-lung-cancer-post-xenium_hd_ffpe/Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2_S1_L001_R1_001.fastq.gz,https://raw.githubusercontent.com/nf-core/test-datasets/spatialvi/testdata/human-lung-cancer-post-xenium_hd_ffpe/Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2_S1_L001_R2_001.fastq.gz,https://raw.githubusercontent.com/nf-core/test-datasets/spatialvi/testdata/human-lung-cancer-post-xenium_hd_ffpe/Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2_tissue_image.btf,https://raw.githubusercontent.com/nf-core/test-datasets/spatialvi/testdata/human-lung-cancer-post-xenium_hd_ffpe/Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2_image.tif,H1-84QJZFR,D1,,,,
+Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2,https://raw.githubusercontent.com/nf-core/test-datasets/spatialvi/testdata/human-lung-cancer-post-xenium_hd_ffpe/Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2_S1_L002_R1_001.fastq.gz,https://raw.githubusercontent.com/nf-core/test-datasets/spatialvi/testdata/human-lung-cancer-post-xenium_hd_ffpe/Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2_S1_L002_R2_001.fastq.gz,https://raw.githubusercontent.com/nf-core/test-datasets/spatialvi/testdata/human-lung-cancer-post-xenium_hd_ffpe/Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2_tissue_image.btf,https://raw.githubusercontent.com/nf-core/test-datasets/spatialvi/testdata/human-lung-cancer-post-xenium_hd_ffpe/Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2_image.tif,H1-84QJZFR,D1,,,,
diff --git a/assets/samplesheet_test_long.csv b/assets/samplesheet_test_long.csv
new file mode 100644
index 0000000..e3c4f7b
--- /dev/null
+++ b/assets/samplesheet_test_long.csv
@@ -0,0 +1,2 @@
+sample,fastq_1,fastq_2,image,cytaimage,slide,area,darkimage,colorizedimage,manual_alignment,slidefile
+Visium_HD_Human_Colon_Cancer,https://raw.githubusercontent.com/nfdata-omics/test-datasets/spatialomics/Visium_HD_Human_Colon_Cancer/Visium_HD_Human_Colon_Cancer_chr21_sub40000_P2_S2_L001_R1_001.fastq.gz,https://raw.githubusercontent.com/nfdata-omics/test-datasets/spatialomics/Visium_HD_Human_Colon_Cancer/Visium_HD_Human_Colon_Cancer_chr21_sub40000_P2_S2_L001_R2_001.fastq.gz,,https://raw.githubusercontent.com/nfdata-omics/test-datasets/spatialomics/Visium_HD_Human_Colon_Cancer/Visium_HD_Human_Colon_Cancer_P2_image.tif,H1-VM2JXXK,A1,,,,
diff --git a/conf/test.config b/conf/test.config
index 51cb49a..268cafb 100644
--- a/conf/test.config
+++ b/conf/test.config
@@ -24,9 +24,7 @@ params {
 
     // Input
     input             = "${projectDir}/assets/samplesheet_test.csv"
-    probeset          = "https://raw.githubusercontent.com/nfdata-omics/test-datasets/spatialomics/Visium_HD_Human_Colon_Cancer/GSM8594568_P2CRC_probe_set.csv"
-    fasta             = "https://raw.githubusercontent.com/nfdata-omics/test-datasets/refs/chr21-cellranger/chr21.fa.gz"
-    gtf               = "https://raw.githubusercontent.com/nfdata-omics/test-datasets/refs/chr21-cellranger/chr21.gtf.gz"
-    reference_name    = "GRCh38"
+    probeset          = "https://raw.githubusercontent.com/nf-core/test-datasets/spatialvi/testdata/human-lung-cancer-post-xenium_hd_ffpe/Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2_probe_set.csv"
+    spaceranger_index = "https://raw.githubusercontent.com/nf-core/test-datasets/spatialvi/testdata/GRCh38.tar.gz"
 
 }
diff --git a/conf/test_long.config b/conf/test_long.config
new file mode 100644
index 0000000..f893dc5
--- /dev/null
+++ b/conf/test_long.config
@@ -0,0 +1,32 @@
+/*
+~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
+    Nextflow config file for running minimal tests
+~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
+    Defines input files and everything required to run a fast and simple pipeline test.
+
+    Use as follows:
+        nextflow run nfdata-omics/spatialomics -profile test_long,<docker/singularity> --outdir <OUTDIR>
+
+----------------------------------------------------------------------------------------
+*/
+
+process {
+    resourceLimits = [
+        cpus: 2,
+        memory: '15.GB',
+        time: '1.h'
+    ]
+}
+
+params {
+    config_profile_name        = 'Long Test profile'
+    config_profile_description = 'Minimal test dataset to check pipeline function, with full chromosome and ref building'
+
+    // Input
+    input             = "${projectDir}/assets/samplesheet_test_long.csv"
+    probeset          = "https://raw.githubusercontent.com/nfdata-omics/test-datasets/spatialomics/Visium_HD_Human_Colon_Cancer/GSM8594568_P2CRC_probe_set.csv"
+    fasta             = "https://raw.githubusercontent.com/nfdata-omics/test-datasets/refs/chr21-cellranger/chr21.fa.gz"
+    gtf               = "https://raw.githubusercontent.com/nfdata-omics/test-datasets/refs/chr21-cellranger/chr21.gtf.gz"
+    reference_name    = "GRCh38"
+
+}
diff --git a/nextflow.config b/nextflow.config
index b21db1b..8b3b04d 100644
--- a/nextflow.config
+++ b/nextflow.config
@@ -169,6 +169,7 @@ profiles {
         singularity.runOptions  = '--nv'
     }
     test      { includeConfig 'conf/test.config'      }
+    test_long { includeConfig 'conf/test_long.config' }
     test_full { includeConfig 'conf/test_full.config' }
 }
 

From 83d29da814a514e2c0b0f06755dc05a633f4ac8d Mon Sep 17 00:00:00 2001
From: Matteo Bonfanti <matteo.bonfanti@fht.org>
Date: Tue, 4 Nov 2025 12:15:47 +0100
Subject: [PATCH 09/18] fix when reference provided as tar

---
 main.nf                                | 2 +-
 subworkflows/local/prepare_ref/main.nf | 2 +-
 2 files changed, 2 insertions(+), 2 deletions(-)

diff --git a/main.nf b/main.nf
index d19c6b9..7c86ada 100644
--- a/main.nf
+++ b/main.nf
@@ -48,7 +48,7 @@ workflow NFDATAOMICS_SPATIALOMICS {
     ch_fasta              = params.fasta             ? Channel.value(file(params.fasta, checkIfExists: true))             : Channel.empty()
     ch_gtf                = params.gtf               ? Channel.value(file(params.gtf, checkIfExists: true))               : Channel.empty()
     ch_gff                = params.gff               ? Channel.value(file(params.gff, checkIfExists: true))               : Channel.empty()
-    ch_spaceranger_index  = params.spaceranger_index ? Channel.value(file(params.spaceranger_index, checkIfExists: true)) : Channel.empty()
+    ch_spaceranger_index  = params.spaceranger_index ? file(params.spaceranger_index, checkIfExists: true) : Channel.empty()
     ch_probeset           = params.probeset          ? Channel.value(file(params.probeset, checkIfExists: true))          : Channel.empty()
 
     //
diff --git a/subworkflows/local/prepare_ref/main.nf b/subworkflows/local/prepare_ref/main.nf
index fc0daad..32edef5 100644
--- a/subworkflows/local/prepare_ref/main.nf
+++ b/subworkflows/local/prepare_ref/main.nf
@@ -39,7 +39,7 @@ workflow PREPARE_REF {
         // Define spaceranger index channel from the user-provided one
         if (params.spaceranger_index ==~ /.*\.tar\.gz$/) {
             UNTAR_SPACERANGER_REF ([
-                ["id": file(params.spaceranger_index).name.replaceAll(/\.(tar)(\.gz)?$/, '')],
+                ["id": params.spaceranger_index.tokenize('/').last().replaceAll(/\.(tar)(\.gz)?$/, '')],
                 spaceranger_index
             ])
             ch_spaceranger_index = UNTAR_SPACERANGER_REF.out.untar.map{ it[1] }

From 396945bc774434ed857a946b2692a41ddea6cbcf Mon Sep 17 00:00:00 2001
From: Matteo Bonfanti <matteo.bonfanti@fht.org>
Date: Tue, 4 Nov 2025 12:16:10 +0100
Subject: [PATCH 10/18] added snapshot for the default test

---
 tests/default.nf.test.snap | 370 ++++++++++++++++++-------------------
 1 file changed, 180 insertions(+), 190 deletions(-)

diff --git a/tests/default.nf.test.snap b/tests/default.nf.test.snap
index eb1a0b7..a0549ec 100644
--- a/tests/default.nf.test.snap
+++ b/tests/default.nf.test.snap
@@ -11,20 +11,11 @@
                 "FQ_LINT": {
                     "fq": "0.12.0 (2024-07-08)"
                 },
-                "GUNZIP_FASTA": {
-                    "gunzip": 1.13
-                },
-                "GUNZIP_GTF": {
-                    "gunzip": 1.13
-                },
                 "SPACERANGER_COUNT": {
                     "spaceranger": "3.1.3"
                 },
-                "SPACERANGER_MKGTF": {
-                    "spaceranger": "3.1.3"
-                },
-                "SPACERANGER_MKREF": {
-                    "spaceranger": "3.1.3"
+                "UNTAR_SPACERANGER_REF": {
+                    "untar": 1.34
                 },
                 "Workflow": {
                     "nfdata-omics/spatialomics": "v0.0.1dev"
@@ -49,6 +40,7 @@
                 "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/count/outs/binned_outputs/square_002um/raw_probe_bc_matrix.h5",
                 "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/count/outs/binned_outputs/square_002um/spatial",
                 "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/count/outs/binned_outputs/square_002um/spatial/aligned_fiducials.jpg",
+                "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/count/outs/binned_outputs/square_002um/spatial/aligned_tissue_image.jpg",
                 "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/count/outs/binned_outputs/square_002um/spatial/cytassist_image.tiff",
                 "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/count/outs/binned_outputs/square_002um/spatial/detected_tissue_image.jpg",
                 "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/count/outs/binned_outputs/square_002um/spatial/scalefactors_json.json",
@@ -122,6 +114,7 @@
                 "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/count/outs/binned_outputs/square_008um/raw_feature_bc_matrix/matrix.mtx.gz",
                 "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/count/outs/binned_outputs/square_008um/spatial",
                 "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/count/outs/binned_outputs/square_008um/spatial/aligned_fiducials.jpg",
+                "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/count/outs/binned_outputs/square_008um/spatial/aligned_tissue_image.jpg",
                 "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/count/outs/binned_outputs/square_008um/spatial/cytassist_image.tiff",
                 "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/count/outs/binned_outputs/square_008um/spatial/detected_tissue_image.jpg",
                 "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/count/outs/binned_outputs/square_008um/spatial/scalefactors_json.json",
@@ -194,6 +187,7 @@
                 "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/count/outs/binned_outputs/square_016um/raw_feature_bc_matrix/matrix.mtx.gz",
                 "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/count/outs/binned_outputs/square_016um/spatial",
                 "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/count/outs/binned_outputs/square_016um/spatial/aligned_fiducials.jpg",
+                "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/count/outs/binned_outputs/square_016um/spatial/aligned_tissue_image.jpg",
                 "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/count/outs/binned_outputs/square_016um/spatial/cytassist_image.tiff",
                 "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/count/outs/binned_outputs/square_016um/spatial/detected_tissue_image.jpg",
                 "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/count/outs/binned_outputs/square_016um/spatial/scalefactors_json.json",
@@ -209,6 +203,7 @@
                 "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/count/outs/probe_set.csv",
                 "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/count/outs/spatial",
                 "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/count/outs/spatial/aligned_fiducials.jpg",
+                "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/count/outs/spatial/aligned_tissue_image.jpg",
                 "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/count/outs/spatial/cytassist_image.tiff",
                 "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/count/outs/spatial/detected_tissue_image.jpg",
                 "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/count/outs/spatial/tissue_hires_image.png",
@@ -224,9 +219,6 @@
                 "fastqc/Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2_2_fastqc.zip",
                 "fq",
                 "fq/Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2.fq_lint.txt",
-                "gunzip",
-                "gunzip/chr21.fa",
-                "gunzip/chr21.gtf",
                 "multiqc",
                 "multiqc/multiqc_data",
                 "multiqc/multiqc_data/fastqc-status-check-heatmap.txt",
@@ -288,190 +280,188 @@
                 "multiqc/multiqc_report.html",
                 "pipeline_info",
                 "pipeline_info/spatialomics_software_mqc_versions.yml",
-                "reference",
-                "reference/mkgtf",
-                "reference/mkgtf/chr21.filtered.gtf",
-                "reference/mkref",
-                "reference/mkref/GRCh38",
-                "reference/mkref/GRCh38/fasta",
-                "reference/mkref/GRCh38/fasta/genome.fa",
-                "reference/mkref/GRCh38/fasta/genome.fa.fai",
-                "reference/mkref/GRCh38/genes",
-                "reference/mkref/GRCh38/genes/genes.gtf.gz",
-                "reference/mkref/GRCh38/reference.json",
-                "reference/mkref/GRCh38/star",
-                "reference/mkref/GRCh38/star/Genome",
-                "reference/mkref/GRCh38/star/SA",
-                "reference/mkref/GRCh38/star/SAindex",
-                "reference/mkref/GRCh38/star/chrLength.txt",
-                "reference/mkref/GRCh38/star/chrName.txt",
-                "reference/mkref/GRCh38/star/chrNameLength.txt",
-                "reference/mkref/GRCh38/star/chrStart.txt",
-                "reference/mkref/GRCh38/star/exonGeTrInfo.tab",
-                "reference/mkref/GRCh38/star/exonInfo.tab",
-                "reference/mkref/GRCh38/star/geneInfo.tab",
-                "reference/mkref/GRCh38/star/genomeParameters.txt",
-                "reference/mkref/GRCh38/star/sjdbInfo.txt",
-                "reference/mkref/GRCh38/star/sjdbList.fromGTF.out.tab",
-                "reference/mkref/GRCh38/star/sjdbList.out.tab",
-                "reference/mkref/GRCh38/star/transcriptInfo.tab"
+                "untar",
+                "untar/GRCh38",
+                "untar/GRCh38/fasta",
+                "untar/GRCh38/fasta/genome.fa",
+                "untar/GRCh38/fasta/genome.fa.fai",
+                "untar/GRCh38/genes",
+                "untar/GRCh38/genes/genes.gtf.gz",
+                "untar/GRCh38/reference.json",
+                "untar/GRCh38/star",
+                "untar/GRCh38/star/Genome",
+                "untar/GRCh38/star/SA",
+                "untar/GRCh38/star/SAindex",
+                "untar/GRCh38/star/chrLength.txt",
+                "untar/GRCh38/star/chrName.txt",
+                "untar/GRCh38/star/chrNameLength.txt",
+                "untar/GRCh38/star/chrStart.txt",
+                "untar/GRCh38/star/exonGeTrInfo.tab",
+                "untar/GRCh38/star/exonInfo.tab",
+                "untar/GRCh38/star/geneInfo.tab",
+                "untar/GRCh38/star/genomeParameters.txt",
+                "untar/GRCh38/star/sjdbInfo.txt",
+                "untar/GRCh38/star/sjdbList.fromGTF.out.tab",
+                "untar/GRCh38/star/sjdbList.out.tab",
+                "untar/GRCh38/star/transcriptInfo.tab"
             ],
             [
-                "filtered_feature_bc_matrix.h5:md5,4dd78df810f819a38d52c5b26be4359e",
-                "barcodes.tsv.gz:md5,0de501f1b0116db710b76671732f388e",
-                "features.tsv.gz:md5,94bb77b14dffd444bfcffd828c84e677",
-                "matrix.mtx.gz:md5,a13354c6dbceb8f9c2b6330a4cfefe02",
-                "raw_feature_bc_matrix.h5:md5,d232789c8fb5dedec327a5fd03b1e0bd",
-                "barcodes.tsv.gz:md5,eb4db44b78def716639651a69cd07d33",
-                "features.tsv.gz:md5,4ab9e3b29c73296728a6525a28660704",
-                "matrix.mtx.gz:md5,65d58b5987279592355861276233a096",
-                "raw_probe_bc_matrix.h5:md5,1a464bd4efc8f8a3554fbc3953e3becc",
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-                "fastqc_sequence_duplication_levels_plot.txt:md5,79c07fa860890c611607352d89d64ea2",
+                "aligned_fiducials.jpg:md5,edde03debc796feed00857c268c6f917",
+                "aligned_tissue_image.jpg:md5,aa844ffd606a7070c5d0de847c723b76",
+                "cytassist_image.tiff:md5,1328dd8ea02922375719f38e3de516ca",
+                "detected_tissue_image.jpg:md5,d4ba1f337bbf9a6d4028139b8acb2411",
+                "tissue_hires_image.png:md5,e86177781b07119c5a680ea4af1d1d0d",
+                "tissue_lowres_image.png:md5,9aad2a56128c450c76fabbb44fb4c85a",
+                "web_summary.html:md5,c319a8dc7b2f4ddda795f4ac23b414db",
+                "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2_1.merged.fastq.gz:md5,ffa0655a818f9c2e1a0d7bc7aa789d56",
+                "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2_2.merged.fastq.gz:md5,23d07d8f5333d96fed828203c2d1c840",
+                "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2.fq_lint.txt:md5,9d29644c14ccc39eb5dde2d6d6b66398",
+                "fastqc-status-check-heatmap.txt:md5,4310b189f786b63e554e1db93160a108",
+                "fastqc_overrepresented_sequences_plot.txt:md5,29526e23692ce67f210bebbbc649b78c",
+                "fastqc_per_base_n_content_plot.txt:md5,60c30869b5226368bf9e42881e1f06b8",
+                "fastqc_per_base_sequence_quality_plot.txt:md5,4fdbdc0489bca53bdf3463a7d47154e6",
+                "fastqc_per_sequence_gc_content_plot_Counts.txt:md5,6c38ba4c544c7f71f5290c30359658b7",
+                "fastqc_per_sequence_gc_content_plot_Percentages.txt:md5,1e4db0b910084bffea76ddd16d5935b5",
+                "fastqc_per_sequence_quality_scores_plot.txt:md5,d5e2d3fe59e5ef3f3664febe74e3549d",
+                "fastqc_sequence_counts_plot.txt:md5,19494d2928422e419de7a9ea447327f8",
+                "fastqc_sequence_duplication_levels_plot.txt:md5,b0bb0df056d4a31dcaec480e98d94939",
                 "multiqc_citations.txt:md5,4c806e63a283ec1b7e78cdae3a923d4f",
-                "multiqc_fastqc.txt:md5,814e07df7cca15a49026ba5af4a25e56",
-                "multiqc_general_stats.txt:md5,10d8597764dcb14f7b28d70811effeb2",
-                "chr21.filtered.gtf:md5,d2db40ac04bf0bce710a304139ca297b",
-                "genome.fa:md5,a7a8ee17d735d23160137c7793f9f872",
-                "genome.fa.fai:md5,a7e52efeee796ff94ede575403a31586",
-                "genes.gtf.gz:md5,d2db40ac04bf0bce710a304139ca297b",
-                "reference.json:md5,5b5a497cd6bd824b68e305176e9f7625",
-                "Genome:md5,ddad1b8708d0fe4624f0d945f1e53381",
-                "SA:md5,f01f7cd63c62a6b69ad49a9919653a9a",
-                "SAindex:md5,fd7b706c62781e92b759d5e8aa5151a7",
-                "chrLength.txt:md5,b0be0a56ddefa84552742c72d4859eac",
-                "chrName.txt:md5,e99d7d1051eee43ceab5563c2d09fcee",
-                "chrNameLength.txt:md5,c985a141685e8431ec27c782816cb744",
-                "chrStart.txt:md5,6925b594ea2eeb964ba87cd6d42ab98f",
-                "exonGeTrInfo.tab:md5,19506a649344a3dbb33bfa47d794851b",
-                "exonInfo.tab:md5,30350358691756b50407c0f8cb6ce5b3",
-                "geneInfo.tab:md5,67cb5583677c9b6f4c5490c6a89dba87",
-                "genomeParameters.txt:md5,195a73612305560d9d2c68c046518727",
-                "sjdbInfo.txt:md5,a544cc53ebf721c5d55d1995abcc907e",
-                "sjdbList.fromGTF.out.tab:md5,3427299e4047e7ba0404e07fb0ac03ee",
-                "sjdbList.out.tab:md5,874227af46e44a89ec5fdbe25d3154a5",
-                "transcriptInfo.tab:md5,a86211e41ef8fd5bcce871ebbbe39638"
+                "multiqc_fastqc.txt:md5,6c8ca51029ac53d1eb6d9ea10817f8bc",
+                "multiqc_general_stats.txt:md5,cf51b986de00e0ef3a35b82d16c5e504",
+                "genome.fa:md5,f315020d899597c1b57e5fe9f60f4c3e",
+                "genome.fa.fai:md5,3520cd30e1b100e55f578db9c855f685",
+                "genes.gtf.gz:md5,50fc877b1c53b36b3b413aff88bda48c",
+                "reference.json:md5,0574f4395bb98647a69feccde59fc656",
+                "Genome:md5,22102926fadf5890e905ca71b2da3f35",
+                "SA:md5,bcf3e1a855783105150b46c905465333",
+                "SAindex:md5,b93fb07d342e6c32a00ebc4311c0ad38",
+                "chrLength.txt:md5,c81f40f27e72606d7d07097c1d56a5b5",
+                "chrName.txt:md5,5ae68a67b70976ee95342a7451cb5af1",
+                "chrNameLength.txt:md5,b190587cae0531f3cf25552d8aa674db",
+                "chrStart.txt:md5,bc73df776dd3d5bb9cfcbcba60880519",
+                "exonGeTrInfo.tab:md5,72d4dd88d25e3c5b0bb72b12b4ac99a8",
+                "exonInfo.tab:md5,0d560290fab688b7268d88d5494bf9fe",
+                "geneInfo.tab:md5,8b608537307443ffaee4927d2b428805",
+                "genomeParameters.txt:md5,a5ca08289b86e0d906683587cbc1049c",
+                "sjdbInfo.txt:md5,5690ea9d9f09f7ff85b7fd47bd234903",
+                "sjdbList.fromGTF.out.tab:md5,8760c33e966dad0b39f440301ebbdee4",
+                "sjdbList.out.tab:md5,9e4f991abbbfeb3935a2bb21b9e258f1",
+                "transcriptInfo.tab:md5,0c3a5adb49d15e5feff81db8e29f2e36"
             ]
         ],
         "meta": {
             "nf-test": "0.9.2",
-            "nextflow": "25.04.8"
+            "nextflow": "25.04.3"
         },
-        "timestamp": "2025-11-03T15:37:00.437153276"
+        "timestamp": "2025-11-04T12:06:58.900086215"
     }
 }
\ No newline at end of file

From eecb71166a7f7d4c15a9ae882566e315022e86df Mon Sep 17 00:00:00 2001
From: Matteo Bonfanti <matteo.bonfanti@fht.org>
Date: Tue, 4 Nov 2025 13:24:10 +0100
Subject: [PATCH 11/18] Increase resource limits for CPUs and memory

---
 conf/test.config | 4 ++--
 1 file changed, 2 insertions(+), 2 deletions(-)

diff --git a/conf/test.config b/conf/test.config
index 268cafb..8d049f3 100644
--- a/conf/test.config
+++ b/conf/test.config
@@ -12,8 +12,8 @@
 
 process {
     resourceLimits = [
-        cpus: 2,
-        memory: '15.GB',
+        cpus: 4,
+        memory: '16.GB',
         time: '1.h'
     ]
 }

From e4944a6a7b46edbc7c7d2e6f0b66db3fc5e491d3 Mon Sep 17 00:00:00 2001
From: Matteo Bonfanti <matteo.bonfanti@fht.org>
Date: Tue, 4 Nov 2025 13:26:20 +0100
Subject: [PATCH 12/18] Reduce memory limit from 16GB to 12GB

---
 conf/test.config | 2 +-
 1 file changed, 1 insertion(+), 1 deletion(-)

diff --git a/conf/test.config b/conf/test.config
index 8d049f3..00e70c1 100644
--- a/conf/test.config
+++ b/conf/test.config
@@ -13,7 +13,7 @@
 process {
     resourceLimits = [
         cpus: 4,
-        memory: '16.GB',
+        memory: '12.GB',
         time: '1.h'
     ]
 }

From 158593c3cff7e9a7b621daea8fea050b1ca614bb Mon Sep 17 00:00:00 2001
From: Matteo Bonfanti <matteo.bonfanti@fht.org>
Date: Tue, 4 Nov 2025 14:08:21 +0100
Subject: [PATCH 13/18] fixing output organization

---
 conf/modules.config | 61 +++++++++++++++++++++++++++++++++++++++++----
 1 file changed, 56 insertions(+), 5 deletions(-)

diff --git a/conf/modules.config b/conf/modules.config
index 3df6b91..ba3c64f 100644
--- a/conf/modules.config
+++ b/conf/modules.config
@@ -18,8 +18,34 @@ process {
         saveAs: { filename -> filename.equals('versions.yml') ? null : filename }
     ]
 
-    withName: FASTQC {
-        ext.args = '--quiet'
+    withName: 'GUNZIP_FASTA' {
+        publishDir = [
+            enabled: false
+        ]
+    }
+
+    withName: 'GUNZIP_GTF' {
+        publishDir = [
+            enabled: false
+        ]
+    }
+
+    withName: 'GUNZIP_GFF' {
+        publishDir = [
+            enabled: false
+        ]
+    }
+
+    withName: 'UNTAR_SPACERANGER_REF' {
+        publishDir = [
+            enabled: false
+        ]
+    }
+
+    withName: 'GFFREAD' {
+        publishDir = [
+            enabled: false
+        ]
     }
 
     withName: SPACERANGER_MKGTF {
@@ -55,11 +81,36 @@ process {
         ]
     }
 
+    withName: 'CAT_FASTQ' {
+        publishDir = [
+            enabled: false
+        ]
+    }
+
+    withName: 'FQ_LINT' {
+        publishDir = [
+            enabled: false
+        ]
+    }
+
+    withName: FASTQC {
+        ext.args = '--quiet'
+    }
+
     withName: SPACERANGER_COUNT {
         publishDir = [
-            path: { "${params.outdir}/${meta.id}/count" },
-            mode: params.publish_dir_mode,
-            saveAs: { filename -> filename.equals('versions.yml') ? null : filename }
+            [
+                path: { "${params.outdir}/count/" },
+                mode: params.publish_dir_mode,
+                pattern: "outs/web_summary.html",
+                saveAs: { "${meta.id}_web_summary.html" }
+            ],
+            [
+                path: { "${params.outdir}/count/${meta.id}" },
+                mode: params.publish_dir_mode,
+                pattern: "outs/*",
+                saveAs: { filename.split('/')[1] }
+            ]
         ]
         ext.args   = "--create-bam true"
         time   = { 240.h * task.attempt }

From 0ee8d6de60f9ef1a27a3230a425b33c7d9f6e14f Mon Sep 17 00:00:00 2001
From: Matteo Bonfanti <matteo.bonfanti@fht.org>
Date: Tue, 4 Nov 2025 16:09:07 +0100
Subject: [PATCH 14/18] fix closure in SPACERANGER_COUNT

---
 conf/modules.config | 2 +-
 1 file changed, 1 insertion(+), 1 deletion(-)

diff --git a/conf/modules.config b/conf/modules.config
index ba3c64f..1727ba4 100644
--- a/conf/modules.config
+++ b/conf/modules.config
@@ -109,7 +109,7 @@ process {
                 path: { "${params.outdir}/count/${meta.id}" },
                 mode: params.publish_dir_mode,
                 pattern: "outs/*",
-                saveAs: { filename.split('/')[1] }
+                saveAs: { filename -> filename.split('/')[1] }
             ]
         ]
         ext.args   = "--create-bam true"

From fb8e04a8b65d25a23397d5831e49126b9276da87 Mon Sep 17 00:00:00 2001
From: Matteo Bonfanti <matteo.bonfanti@fht.org>
Date: Tue, 4 Nov 2025 16:09:26 +0100
Subject: [PATCH 15/18] unstable output from cellranger excluded from test

---
 tests/.nftignore | 3 +++
 1 file changed, 3 insertions(+)

diff --git a/tests/.nftignore b/tests/.nftignore
index e128a12..c88901e 100644
--- a/tests/.nftignore
+++ b/tests/.nftignore
@@ -10,3 +10,6 @@ multiqc/multiqc_plots/{svg,pdf,png}/*.{svg,pdf,png}
 multiqc/multiqc_report.html
 fastqc/*_fastqc.{html,zip}
 pipeline_info/*.{html,json,txt,yml}
+count/*/*.cloupe
+count/*/molecule_info.h5
+count/*web_summary.html

From b887354d0a3fd5c6bee0fde76aeebd1d6abea384 Mon Sep 17 00:00:00 2001
From: Matteo Bonfanti <matteo.bonfanti@fht.org>
Date: Tue, 4 Nov 2025 17:56:15 +0100
Subject: [PATCH 16/18] copying all subfolders from spaceranger output

---
 conf/modules.config | 4 ++--
 1 file changed, 2 insertions(+), 2 deletions(-)

diff --git a/conf/modules.config b/conf/modules.config
index 1727ba4..65816b7 100644
--- a/conf/modules.config
+++ b/conf/modules.config
@@ -108,8 +108,8 @@ process {
             [
                 path: { "${params.outdir}/count/${meta.id}" },
                 mode: params.publish_dir_mode,
-                pattern: "outs/*",
-                saveAs: { filename -> filename.split('/')[1] }
+                pattern: "outs/**",
+                saveAs: { filename -> filename.contains('/') ? filename.substring(filename.indexOf('/') + 1) : filename }
             ]
         ]
         ext.args   = "--create-bam true"

From a987f12a69dfdadc49153add24fb5aff78773555 Mon Sep 17 00:00:00 2001
From: Matteo Bonfanti <matteo.bonfanti@fht.org>
Date: Tue, 4 Nov 2025 19:37:12 +0100
Subject: [PATCH 17/18] removed loupe files from test

---
 tests/.nftignore           |   2 +-
 tests/default.nf.test.snap | 436 ++++++++++++++++---------------------
 2 files changed, 192 insertions(+), 246 deletions(-)

diff --git a/tests/.nftignore b/tests/.nftignore
index c88901e..b15cfe6 100644
--- a/tests/.nftignore
+++ b/tests/.nftignore
@@ -10,6 +10,6 @@ multiqc/multiqc_plots/{svg,pdf,png}/*.{svg,pdf,png}
 multiqc/multiqc_report.html
 fastqc/*_fastqc.{html,zip}
 pipeline_info/*.{html,json,txt,yml}
-count/*/*.cloupe
+count/**.cloupe
 count/*/molecule_info.h5
 count/*web_summary.html
diff --git a/tests/default.nf.test.snap b/tests/default.nf.test.snap
index a0549ec..7ccfeb9 100644
--- a/tests/default.nf.test.snap
+++ b/tests/default.nf.test.snap
@@ -22,203 +22,198 @@
                 }
             },
             [
-                "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2",
-                "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/count",
-                "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/count/outs",
-                "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/count/outs/binned_outputs",
-                "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/count/outs/binned_outputs/square_002um",
-                "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/count/outs/binned_outputs/square_002um/filtered_feature_bc_matrix",
-                "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/count/outs/binned_outputs/square_002um/filtered_feature_bc_matrix.h5",
-                "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/count/outs/binned_outputs/square_002um/filtered_feature_bc_matrix/barcodes.tsv.gz",
-                "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/count/outs/binned_outputs/square_002um/filtered_feature_bc_matrix/features.tsv.gz",
-                "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/count/outs/binned_outputs/square_002um/filtered_feature_bc_matrix/matrix.mtx.gz",
-                "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/count/outs/binned_outputs/square_002um/raw_feature_bc_matrix",
-                "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/count/outs/binned_outputs/square_002um/raw_feature_bc_matrix.h5",
-                "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/count/outs/binned_outputs/square_002um/raw_feature_bc_matrix/barcodes.tsv.gz",
-                "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/count/outs/binned_outputs/square_002um/raw_feature_bc_matrix/features.tsv.gz",
-                "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/count/outs/binned_outputs/square_002um/raw_feature_bc_matrix/matrix.mtx.gz",
-                "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/count/outs/binned_outputs/square_002um/raw_probe_bc_matrix.h5",
-                "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/count/outs/binned_outputs/square_002um/spatial",
-                "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/count/outs/binned_outputs/square_002um/spatial/aligned_fiducials.jpg",
-                "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/count/outs/binned_outputs/square_002um/spatial/aligned_tissue_image.jpg",
-                "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/count/outs/binned_outputs/square_002um/spatial/cytassist_image.tiff",
-                "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/count/outs/binned_outputs/square_002um/spatial/detected_tissue_image.jpg",
-                "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/count/outs/binned_outputs/square_002um/spatial/scalefactors_json.json",
-                "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/count/outs/binned_outputs/square_002um/spatial/tissue_hires_image.png",
-                "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/count/outs/binned_outputs/square_002um/spatial/tissue_lowres_image.png",
-                "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/count/outs/binned_outputs/square_002um/spatial/tissue_positions.parquet",
-                "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/count/outs/binned_outputs/square_008um",
-                "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/count/outs/binned_outputs/square_008um/analysis",
-                "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/count/outs/binned_outputs/square_008um/analysis/clustering",
-                "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/count/outs/binned_outputs/square_008um/analysis/clustering/gene_expression_graphclust",
-                "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/count/outs/binned_outputs/square_008um/analysis/clustering/gene_expression_graphclust/clusters.csv",
-                "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/count/outs/binned_outputs/square_008um/analysis/clustering/gene_expression_kmeans_10_clusters",
-                "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/count/outs/binned_outputs/square_008um/analysis/clustering/gene_expression_kmeans_10_clusters/clusters.csv",
-                "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/count/outs/binned_outputs/square_008um/analysis/clustering/gene_expression_kmeans_2_clusters",
-                "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/count/outs/binned_outputs/square_008um/analysis/clustering/gene_expression_kmeans_2_clusters/clusters.csv",
-                "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/count/outs/binned_outputs/square_008um/analysis/clustering/gene_expression_kmeans_3_clusters",
-                "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/count/outs/binned_outputs/square_008um/analysis/clustering/gene_expression_kmeans_3_clusters/clusters.csv",
-                "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/count/outs/binned_outputs/square_008um/analysis/clustering/gene_expression_kmeans_4_clusters",
-                "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/count/outs/binned_outputs/square_008um/analysis/clustering/gene_expression_kmeans_4_clusters/clusters.csv",
-                "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/count/outs/binned_outputs/square_008um/analysis/clustering/gene_expression_kmeans_5_clusters",
-                "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/count/outs/binned_outputs/square_008um/analysis/clustering/gene_expression_kmeans_5_clusters/clusters.csv",
-                "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/count/outs/binned_outputs/square_008um/analysis/clustering/gene_expression_kmeans_6_clusters",
-                "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/count/outs/binned_outputs/square_008um/analysis/clustering/gene_expression_kmeans_6_clusters/clusters.csv",
-                "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/count/outs/binned_outputs/square_008um/analysis/clustering/gene_expression_kmeans_7_clusters",
-                "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/count/outs/binned_outputs/square_008um/analysis/clustering/gene_expression_kmeans_7_clusters/clusters.csv",
-                "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/count/outs/binned_outputs/square_008um/analysis/clustering/gene_expression_kmeans_8_clusters",
-                "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/count/outs/binned_outputs/square_008um/analysis/clustering/gene_expression_kmeans_8_clusters/clusters.csv",
-                "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/count/outs/binned_outputs/square_008um/analysis/clustering/gene_expression_kmeans_9_clusters",
-                "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/count/outs/binned_outputs/square_008um/analysis/clustering/gene_expression_kmeans_9_clusters/clusters.csv",
-                "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/count/outs/binned_outputs/square_008um/analysis/diffexp",
-                "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/count/outs/binned_outputs/square_008um/analysis/diffexp/gene_expression_graphclust",
-                "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/count/outs/binned_outputs/square_008um/analysis/diffexp/gene_expression_graphclust/differential_expression.csv",
-                "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/count/outs/binned_outputs/square_008um/analysis/diffexp/gene_expression_kmeans_10_clusters",
-                "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/count/outs/binned_outputs/square_008um/analysis/diffexp/gene_expression_kmeans_10_clusters/differential_expression.csv",
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+                "count/Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/binned_outputs/square_016um/spatial/scalefactors_json.json",
+                "count/Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/binned_outputs/square_016um/spatial/tissue_hires_image.png",
+                "count/Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/binned_outputs/square_016um/spatial/tissue_lowres_image.png",
+                "count/Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/binned_outputs/square_016um/spatial/tissue_positions.parquet",
+                "count/Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/cloupe_008um.cloupe",
+                "count/Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/feature_slice.h5",
+                "count/Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/metrics_summary.csv",
+                "count/Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/molecule_info.h5",
+                "count/Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/possorted_genome_bam.bam",
+                "count/Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/possorted_genome_bam.bam.bai",
+                "count/Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/probe_set.csv",
+                "count/Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/spatial",
+                "count/Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/spatial/aligned_fiducials.jpg",
+                "count/Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/spatial/aligned_tissue_image.jpg",
+                "count/Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/spatial/cytassist_image.tiff",
+                "count/Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/spatial/detected_tissue_image.jpg",
+                "count/Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/spatial/tissue_hires_image.png",
+                "count/Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/spatial/tissue_lowres_image.png",
+                "count/Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2/web_summary.html",
+                "count/Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2_web_summary.html",
                 "fastqc",
                 "fastqc/Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2_1_fastqc.html",
                 "fastqc/Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2_1_fastqc.zip",
                 "fastqc/Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2_2_fastqc.html",
                 "fastqc/Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2_2_fastqc.zip",
-                "fq",
-                "fq/Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2.fq_lint.txt",
                 "multiqc",
                 "multiqc/multiqc_data",
                 "multiqc/multiqc_data/fastqc-status-check-heatmap.txt",
@@ -279,31 +274,7 @@
                 "multiqc/multiqc_plots/svg/fastqc_top_overrepresented_sequences_table.svg",
                 "multiqc/multiqc_report.html",
                 "pipeline_info",
-                "pipeline_info/spatialomics_software_mqc_versions.yml",
-                "untar",
-                "untar/GRCh38",
-                "untar/GRCh38/fasta",
-                "untar/GRCh38/fasta/genome.fa",
-                "untar/GRCh38/fasta/genome.fa.fai",
-                "untar/GRCh38/genes",
-                "untar/GRCh38/genes/genes.gtf.gz",
-                "untar/GRCh38/reference.json",
-                "untar/GRCh38/star",
-                "untar/GRCh38/star/Genome",
-                "untar/GRCh38/star/SA",
-                "untar/GRCh38/star/SAindex",
-                "untar/GRCh38/star/chrLength.txt",
-                "untar/GRCh38/star/chrName.txt",
-                "untar/GRCh38/star/chrNameLength.txt",
-                "untar/GRCh38/star/chrStart.txt",
-                "untar/GRCh38/star/exonGeTrInfo.tab",
-                "untar/GRCh38/star/exonInfo.tab",
-                "untar/GRCh38/star/geneInfo.tab",
-                "untar/GRCh38/star/genomeParameters.txt",
-                "untar/GRCh38/star/sjdbInfo.txt",
-                "untar/GRCh38/star/sjdbList.fromGTF.out.tab",
-                "untar/GRCh38/star/sjdbList.out.tab",
-                "untar/GRCh38/star/transcriptInfo.tab"
+                "pipeline_info/spatialomics_software_mqc_versions.yml"
             ],
             [
                 "filtered_feature_bc_matrix.h5:md5,0c04bf2ea059df078bec3e765b1f3105",
@@ -349,7 +320,6 @@
                 "projection.csv:md5,cf8f37dcfe6c718b28a8c13c9eca6dc6",
                 "variance.csv:md5,fc0451575fe03683e19bfc7e1981be3c",
                 "projection.csv:md5,61af621c1e89a2d8d67c74f2d412d548",
-                "cloupe.cloupe:md5,34e3fbea7223136ba13c99c51d7a357f",
                 "filtered_feature_bc_matrix.h5:md5,6363415f0bfdbdae9a3eab5c8d838ae8",
                 "barcodes.tsv.gz:md5,2f707eec36c8c8478071836e375c589d",
                 "features.tsv.gz:md5,53092c021f35473a120886f08871c246",
@@ -408,10 +378,8 @@
                 "tissue_hires_image.png:md5,e86177781b07119c5a680ea4af1d1d0d",
                 "tissue_lowres_image.png:md5,9aad2a56128c450c76fabbb44fb4c85a",
                 "tissue_positions.parquet:md5,36c859a18ff9c86cd1279ac96c433627",
-                "cloupe_008um.cloupe:md5,34e3fbea7223136ba13c99c51d7a357f",
                 "feature_slice.h5:md5,3be11985be80bee6bae0ec7721280c8a",
                 "metrics_summary.csv:md5,bade113e17b62be4e41cd3c98b6e1c81",
-                "molecule_info.h5:md5,a71fd0a61e37b870fb77f1cb4269efdb",
                 "possorted_genome_bam.bam:md5,c9e1bfe554e6762bae55bf733cb9f221",
                 "possorted_genome_bam.bam.bai:md5,76e1653a2f2620e3e6dfb4fee4d4171d",
                 "probe_set.csv:md5,a74f1583689de3c9f95164b2fc090194",
@@ -421,10 +389,7 @@
                 "detected_tissue_image.jpg:md5,d4ba1f337bbf9a6d4028139b8acb2411",
                 "tissue_hires_image.png:md5,e86177781b07119c5a680ea4af1d1d0d",
                 "tissue_lowres_image.png:md5,9aad2a56128c450c76fabbb44fb4c85a",
-                "web_summary.html:md5,c319a8dc7b2f4ddda795f4ac23b414db",
-                "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2_1.merged.fastq.gz:md5,ffa0655a818f9c2e1a0d7bc7aa789d56",
-                "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2_2.merged.fastq.gz:md5,23d07d8f5333d96fed828203c2d1c840",
-                "Visium_HD_Human_Lung_Cancer_HD_Only_Experiment2.fq_lint.txt:md5,9d29644c14ccc39eb5dde2d6d6b66398",
+                "web_summary.html:md5,43a960f3933c57e192bc7087bb9bef0c",
                 "fastqc-status-check-heatmap.txt:md5,4310b189f786b63e554e1db93160a108",
                 "fastqc_overrepresented_sequences_plot.txt:md5,29526e23692ce67f210bebbbc649b78c",
                 "fastqc_per_base_n_content_plot.txt:md5,60c30869b5226368bf9e42881e1f06b8",
@@ -436,32 +401,13 @@
                 "fastqc_sequence_duplication_levels_plot.txt:md5,b0bb0df056d4a31dcaec480e98d94939",
                 "multiqc_citations.txt:md5,4c806e63a283ec1b7e78cdae3a923d4f",
                 "multiqc_fastqc.txt:md5,6c8ca51029ac53d1eb6d9ea10817f8bc",
-                "multiqc_general_stats.txt:md5,cf51b986de00e0ef3a35b82d16c5e504",
-                "genome.fa:md5,f315020d899597c1b57e5fe9f60f4c3e",
-                "genome.fa.fai:md5,3520cd30e1b100e55f578db9c855f685",
-                "genes.gtf.gz:md5,50fc877b1c53b36b3b413aff88bda48c",
-                "reference.json:md5,0574f4395bb98647a69feccde59fc656",
-                "Genome:md5,22102926fadf5890e905ca71b2da3f35",
-                "SA:md5,bcf3e1a855783105150b46c905465333",
-                "SAindex:md5,b93fb07d342e6c32a00ebc4311c0ad38",
-                "chrLength.txt:md5,c81f40f27e72606d7d07097c1d56a5b5",
-                "chrName.txt:md5,5ae68a67b70976ee95342a7451cb5af1",
-                "chrNameLength.txt:md5,b190587cae0531f3cf25552d8aa674db",
-                "chrStart.txt:md5,bc73df776dd3d5bb9cfcbcba60880519",
-                "exonGeTrInfo.tab:md5,72d4dd88d25e3c5b0bb72b12b4ac99a8",
-                "exonInfo.tab:md5,0d560290fab688b7268d88d5494bf9fe",
-                "geneInfo.tab:md5,8b608537307443ffaee4927d2b428805",
-                "genomeParameters.txt:md5,a5ca08289b86e0d906683587cbc1049c",
-                "sjdbInfo.txt:md5,5690ea9d9f09f7ff85b7fd47bd234903",
-                "sjdbList.fromGTF.out.tab:md5,8760c33e966dad0b39f440301ebbdee4",
-                "sjdbList.out.tab:md5,9e4f991abbbfeb3935a2bb21b9e258f1",
-                "transcriptInfo.tab:md5,0c3a5adb49d15e5feff81db8e29f2e36"
+                "multiqc_general_stats.txt:md5,cf51b986de00e0ef3a35b82d16c5e504"
             ]
         ],
         "meta": {
             "nf-test": "0.9.2",
             "nextflow": "25.04.3"
         },
-        "timestamp": "2025-11-04T12:06:58.900086215"
+        "timestamp": "2025-11-04T18:51:17.325765047"
     }
 }
\ No newline at end of file

From 63bb9a44e26898e00faf5109cedb237821945734 Mon Sep 17 00:00:00 2001
From: Matteo Bonfanti <matteo.bonfanti@fht.org>
Date: Wed, 5 Nov 2025 16:38:49 +0100
Subject: [PATCH 18/18] removing web summary from snap

---
 tests/.nftignore           | 2 +-
 tests/default.nf.test.snap | 5 ++---
 2 files changed, 3 insertions(+), 4 deletions(-)

diff --git a/tests/.nftignore b/tests/.nftignore
index b15cfe6..d62a97a 100644
--- a/tests/.nftignore
+++ b/tests/.nftignore
@@ -12,4 +12,4 @@ fastqc/*_fastqc.{html,zip}
 pipeline_info/*.{html,json,txt,yml}
 count/**.cloupe
 count/*/molecule_info.h5
-count/*web_summary.html
+count/**web_summary.html
diff --git a/tests/default.nf.test.snap b/tests/default.nf.test.snap
index 7ccfeb9..782279a 100644
--- a/tests/default.nf.test.snap
+++ b/tests/default.nf.test.snap
@@ -389,7 +389,6 @@
                 "detected_tissue_image.jpg:md5,d4ba1f337bbf9a6d4028139b8acb2411",
                 "tissue_hires_image.png:md5,e86177781b07119c5a680ea4af1d1d0d",
                 "tissue_lowres_image.png:md5,9aad2a56128c450c76fabbb44fb4c85a",
-                "web_summary.html:md5,43a960f3933c57e192bc7087bb9bef0c",
                 "fastqc-status-check-heatmap.txt:md5,4310b189f786b63e554e1db93160a108",
                 "fastqc_overrepresented_sequences_plot.txt:md5,29526e23692ce67f210bebbbc649b78c",
                 "fastqc_per_base_n_content_plot.txt:md5,60c30869b5226368bf9e42881e1f06b8",
@@ -406,8 +405,8 @@
         ],
         "meta": {
             "nf-test": "0.9.2",
-            "nextflow": "25.04.3"
+            "nextflow": "25.04.8"
         },
-        "timestamp": "2025-11-04T18:51:17.325765047"
+        "timestamp": "2025-11-05T15:21:34.914957103"
     }
 }
\ No newline at end of file