ebola-rnaseq

• Galaxy Workflow Data-Flow Brief: Ebola Virus Infection RNA-seq Analysis
• Data-Flow Overview
• Step Details and Collection Operations
• Step 1: `salmon_quant` (Map-over)
• Step 2: `deseq2` (Reduce / Collective modeling)
• Galaxy Tool Equivalents & Unresolved Needs
• Confidence and Open Questions

# Galaxy Workflow Data-Flow Brief: Ebola Virus Infection RNA-seq Analysis

## Data-Flow Overview
The workflow transforms raw paired-end RNA-seq collections into sample-level transcript abundance estimates and downstream gene-level differential expression statistics.

```
[reads_paired_collection (list:paired)] ──┐
                                          ├─► [Step 1: salmon_quant (map-over)] ──► [salmon_quant_collection (list)]
[salmon_index] ───────────────────────────┘                                              │
                                                                                         ▼
[sample_metadata (tabular)] ───────────────────────────────────────────────────────────► [Step 2: deseq2 (reduce)] ──► [deseq2_norm_counts]
                                                                                         ▲                           ──► [deseq2_differential_results]
[tx2gene_map (tabular)] ─────────────────────────────────────────────────────────────────┘                           ──► [deseq2_plots]
```

## Step Details and Collection Operations

### Step 1: `salmon_quant` (Map-over)
- **Operation**: Pseudoalignment and transcript abundance quantification.
- **Input Wiring**:
  - `paired_reads`: Connected to `reads_paired_collection` (mapped over each paired element).
  - `index`: Connected to `salmon_index`.
- **Collection Behavior**: Maps over the input `list:paired` collection, preserving sample element identifiers.
- **Output**: `quant.sf` transcript quantification table per element, producing a `list` collection `salmon_quant_collection`.

### Step 2: `deseq2` (Reduce / Collective modeling)
- **Operation**: Multi-factor differential gene expression modeling using DESeq2.
- **Input Wiring**:
  - `counts_or_abundance`: Connected to `salmon_quant_collection` (accepting the collection of quantification tables).
  - `sample_table`: Connected to `sample_metadata`.
  - `tx2gene`: Connected to `tx2gene_map`.
- **Collection Behavior**: Reduces the collection of sample abundances into collective matrix modeling.
- **Outputs**:
  - `normalized_counts`: Tabular normalized expression values across samples.
  - `differential_expression`: Tabular results with log2 fold-changes, p-values, and adjusted FDR.
  - `qc_plots`: Diagnostic PDF containing PCA and dispersion plots.

## Galaxy Tool Equivalents & Unresolved Needs
- `salmon_quant`: Standard Galaxy tool `toolshed.g2.bx.psu.edu/repos/bgruening/salmon/salmon/1.10.0+galaxy0` (or compatible version).
- `deseq2`: Standard Galaxy tool `toolshed.g2.bx.psu.edu/repos/iuc/deseq2/deseq2/2.11.40.8+galaxy0` (or compatible version).
- No custom wrappers required; standard ToolShed tools fulfill all operations.

## Confidence and Open Questions
- High confidence in map-over on `list:paired` feeding directly into DESeq2's collection-aware input interface or standard tabular aggregation.
- Carried open requirement: Verify DESeq2 parameter interface for tximport/tx2gene integration vs feeding gene-level count tables directly.

• Galaxy Workflow Interface Brief: Ebola Virus Infection RNA-seq Analysis
• Workflow Purpose & Overview
• Workflow Inputs
• Workflow Outputs
• Collection Shapes & Mapping Strategy
• Confidence and Assumptions
• Open Questions & Interface Obligations

# Galaxy Workflow Interface Brief: Ebola Virus Infection RNA-seq Analysis

## Workflow Purpose & Overview
Process paired-end RNA-seq reads from human A549 WT and A549 PTPN13-KO cells infected with Ebola virus or mock at 24 and 48 HPI. The workflow performs pseudoalignment and transcript abundance quantification with Salmon, aggregates abundances to gene-level counts, and conducts multi-factor differential expression analysis with DESeq2.

## Workflow Inputs

| Input Identifier | Label | Type | Format / Collection Shape | Optional / Default | Description |
|---|---|---|---|---|---|
| `reads_paired_collection` | Paired-end RNA-seq reads | `data_collection_input` | `list:paired` (`fastqsanger`, `fastqsanger.gz`) | Required | Dataset collection of paired-end Illumina FASTQ reads across experimental samples |
| `salmon_index` | Salmon reference transcriptome index | `data_input` | `tar`, `directory` (or `fasta`) | Required | Precomputed Salmon index directory/archive for human transcriptome targets |
| `sample_metadata` | Sample metadata / Experimental factors | `data_input` | `tabular` | Required | Tabular design matrix specifying sample IDs, Genotype (WT / KO), Condition (Mock / Ebola), and Timepoint (24 / 48) |
| `tx2gene_map` | Transcript-to-gene mapping table | `data_input` | `tabular` | Required | Two-column tab-delimited mapping of transcript IDs to gene IDs |

## Workflow Outputs

| Output Identifier | Label | Source Step / Producer | Datatype / Collection | Public / Checkpoint | Description |
|---|---|---|---|---|---|
| `salmon_quant_collection` | Salmon quantification tables (`quant.sf`) | Salmon step | `list` of `tabular` | Checkpoint / Public | Per-sample transcript abundance estimates and counts |
| `deseq2_norm_counts` | DESeq2 normalized counts matrix | DESeq2 step | `tabular` | Public | Size-factor normalized gene expression matrix across all samples |
| `deseq2_differential_results` | DESeq2 differential expression results | DESeq2 step | `tabular` | Public | Statistical test statistics (log2FC, p-value, padj/FDR) for tested contrasts |
| `deseq2_plots` | DESeq2 diagnostic plots (PCA & dispersions) | DESeq2 step | `pdf` | Public | Quality control PCA and dispersion diagnostic plots |

## Collection Shapes & Mapping Strategy
- **Reads Collection**: Structured as `list:paired` containing each biological sample as an element mapped to forward and reverse FASTQ datasets.
- **Salmon Execution**: Mapped over the `list:paired` collection, outputting a `list` of `quant.sf` tabular datasets.
- **Aggregation & Modeling**: DESeq2 takes the collection of count/abundance tables together with `sample_metadata` and `tx2gene_map` as collective inputs to model variance and differential expression across the 8 experimental groups (2 genotypes × 2 conditions × 2 timepoints).

## Confidence and Assumptions
- High confidence in `list:paired` collection shape for paired-end Illumina RNA-seq.
- High confidence in Salmon + DESeq2 toolchain as standard, well-supported Galaxy ecosystem tools.
- Checkpoint output `salmon_quant_collection` allows isolated verification of the quantification tier before differential modeling.

## Open Questions & Interface Obligations
1. Whether Salmon index is provided as a pre-built index directory/tarball or built dynamically from a FASTA file.
2. The specific DESeq2 design formula (additive `~ Genotype + Time + Condition` vs full interaction `~ Genotype * Time * Condition`).

• Free-form Summary: Ebola Virus Infection RNA-seq Analysis (A549 WT vs PTPN13-KO)
• Workflow Intent
• Methods / Algorithms
• Tools
• Inputs
• Outputs
• Parameters
• Data Availability
• Constraints
• Confidence and Open Questions

# Free-form Summary: Ebola Virus Infection RNA-seq Analysis (A549 WT vs PTPN13-KO)

## Workflow Intent
Perform transcriptomic profiling and differential gene expression analysis of human cell lines (A549 wild-type vs. A549 PTPN13 knockout) infected with Ebola virus versus mock infection across two post-infection time points (24 and 48 HPI). The primary objective is to assess the transcriptional host response, the effect of PTPN13 loss on viral infection kinetics and immune signalling, and time-dependent expression dynamics.

## Methods / Algorithms
1. **Transcript Quantification**: Pseudoalignment and transcript abundance estimation of paired-end RNA-seq reads against human reference transcriptome targets using Salmon.
2. **Gene-Level Summarization / Aggregation**: Mapping transcript-level estimates to gene-level counts and abundances for downstream statistical modeling.
3. **Differential Expression Analysis**: Multi-factor negative binomial generalized linear modeling (DESeq2) testing contrasts across Genotype (WT vs. PTPN13-KO), Infection Condition (Mock vs. Ebola virus), and Time Point (24 HPI vs. 48 HPI).

## Tools
- **Salmon**: Paired-end read pseudoalignment and quantification (`salmon quant`).
- **DESeq2**: Differential gene expression analysis and normalization from quantified counts.
- *(Optional / Downstream helper)*: Gene-to-transcript mapping utility or tximport if converting transcript abundance to gene counts before DESeq2.

## Inputs
- **RNA-seq Reads**: Paired-end Illumina FASTQ reads formatted as a paired dataset collection or list of pairs (`list:paired`):
  - Cell lines: A549 WT and A549 PTPN13-KO
  - Treatment: Mock vs. Ebola virus infection
  - Time points: 24 HPI, 48 HPI
- **Reference Transcriptome / Index**: Human transcriptome sequences (FASTA) or precomputed Salmon index (GRCh38 / Ensembl / GENCODE).
- **Sample Metadata / Design Matrix**: Tabular factor annotations mapping sample identifiers to Genotype (WT / KO), Condition (Mock / Ebola), and Timepoint (24 / 48).
- **Gene-Transcript Mapping Table**: Two-column tabular mapping transcript IDs to gene symbols/IDs for DESeq2 gene aggregation.

## Outputs
- **Transcript & Gene Quantifications**: Salmon quant directories / abundance tabular files (`quant.sf`) per sample.
- **Normalized Counts**: Size-factor normalized expression matrix from DESeq2.
- **Differential Expression Results**: Tabular tables of log2 fold changes, p-values, and adjusted p-values (FDR) for key comparisons (e.g., Ebola vs Mock in WT, Ebola vs Mock in KO, Genotype effect upon infection).
- **Diagnostic / QC Plots**: PCA plot of samples, dispersion estimates, and MA plots generated by DESeq2.

## Parameters
- Paired-end library orientation and automated strandedness detection (`-l A` / automated).
- Default DESeq2 Wald test or LRT (likelihood ratio test) for multi-factor time series designs.
- Significance thresholds typically FDR < 0.05 and |log2FC| > 1.

## Data Availability
- User has experimental data; minimal synthetic/subsampled test fixtures (small FASTQ pairs + mini transcriptome index + sample sheet) will be resolved for automated workflow validation and testing.

## Constraints
- Sequencing read format is paired-end Illumina.
- Organism focus: Human host transcriptome (A549 lung carcinoma line).
- Non-relevant noise terms ("PPR", "DC") mentioned during initial prompt were clarified as conversational noise and excluded.

## Confidence and Open Questions
- **Confidence**: High confidence on paired-end read structure, Salmon quantification, human host reference, and DESeq2 differential calling across the 2x2x2 experimental matrix.
- **Open Questions**:
  1. *Transcriptome vs Dual-Index*: Does the Salmon index contain only the human transcriptome, or is it a combined host + Ebola virus transcriptome to concurrently quantify viral RNA load?
  2. *Design Formula*: Is the primary contrast modeled with interaction terms (e.g. `~ Genotype + Time + Condition + Genotype:Condition`) or as grouped factor contrasts?
  3. *Gene Mapping*: Will the workflow receive a separate tx2gene tabular file, or use built-in tool features / annotations?

plan_version: "1"
source:
  kind: freeform
  name: "Ebola Virus Infection RNA-seq Analysis (A549 WT vs PTPN13-KO)"
  derived_from: intent
  notes: "Synthesized from user interview intent and design briefs for paired-end RNA-seq with Salmon and DESeq2."
workflow:
  title: "Ebola Virus Infection RNA-seq Analysis (A549 WT vs PTPN13-KO)"
  label_source: draft
  notes: "Input and output labels match concrete galaxy-workflow.gxwf.yml."
test_cases:
  - id: test_ebola_rnaseq_pe
    doc: "Execute Salmon quantification on paired reads and run DESeq2 differential analysis across conditions."
    derived_from: intent
    provenance: "Interview intent and IWC transcriptomics test fixture conventions."
    job_inputs:
      - workflow_label: reads_paired_collection
        label_status: resolved
        description: "Collection of paired-end FASTQ reads across experimental samples."
        collection_shape: "list:paired"
        datatype: fastqsanger.gz
        fixture:
          storage: remote-url
          location: "https://zenodo.org/records/13987631/files/SRR5085167_forward.fastqsanger.gz"
          checksum: "SHA-1:f910a2a7764249b690e28d8dcf4d7097d3c533f6"
          provenance: "IWC rnaseq-pe Zenodo record 13987631"
      - workflow_label: salmon_index
        label_status: resolved
        description: "Reference transcriptome FASTA sequence for Salmon indexing."
        collection_shape: null
        datatype: fasta
        fixture:
          storage: remote-url
          location: "https://zenodo.org/records/13987631/files/Saccharomyces_cerevisiae.R64-1-1.cdna.all.fa"
          checksum: null
          provenance: "IWC transcriptomics cDNA FASTA"
      - workflow_label: sample_metadata
        label_status: resolved
        description: "Tabular sample metadata specifying factors for DESeq2."
        collection_shape: null
        datatype: tabular
        fixture:
          storage: in-repo
          location: "test-data/sample_metadata.tabular"
          checksum: null
          provenance: "Synthesized sample annotation matching FASTQ identifiers"
      - workflow_label: tx2gene_map
        label_status: resolved
        description: "Tabular mapping between transcript IDs and gene IDs."
        collection_shape: null
        datatype: tabular
        fixture:
          storage: in-repo
          location: "test-data/tx2gene.tabular"
          checksum: null
          provenance: "Synthesized tx2gene table for transcript aggregation"
    expected_outputs:
      - workflow_label: salmon_quant_collection
        label_status: resolved
        description: "Collection of per-sample Salmon transcript quantification tables."
        output_kind: collection
        collection_shape: list
        assertion_intent:
          - family: has_n_lines
            intent: "Each quant.sf table contains quantified transcript rows."
            expected_value: 10
            tolerance:
              kind: delta
              magnitude: 50
              rationale: "Depends on fixture transcriptome size"
            element_identifier: SRR5085167
            evidence: intent
            confidence: medium
      - workflow_label: deseq2_norm_counts
        label_status: resolved
        description: "Size-factor normalized counts table from DESeq2."
        output_kind: dataset
        collection_shape: null
        assertion_intent:
          - family: has_n_columns
            intent: "Normalized count matrix contains sample columns."
            expected_value: 3
            tolerance: null
            element_identifier: null
            evidence: intent
            confidence: medium
      - workflow_label: deseq2_differential_results
        label_status: resolved
        description: "DESeq2 statistical test results table."
        output_kind: dataset
        collection_shape: null
        assertion_intent:
          - family: has_text
            intent: "Output contains expected DESeq2 header columns (baseMean, log2FoldChange, pvalue, padj)."
            expected_value: "baseMean"
            tolerance: null
            element_identifier: null
            evidence: intent
            confidence: high
      - workflow_label: deseq2_plots
        label_status: resolved
        description: "DESeq2 diagnostic PCA and dispersion plots."
        output_kind: dataset
        collection_shape: null
        assertion_intent:
          - family: has_size
            intent: "Valid diagnostic PDF is generated with non-zero size."
            expected_value: 5000
            tolerance:
              kind: delta
              magnitude: 500000
              rationale: "PDF binary size varies with R graphics device"
            element_identifier: null
            evidence: intent
            confidence: medium
unresolved: []
omissions:
  - target: deseq2_plots
    reason: "Exact pixel or stream equality of R graphics is non-deterministic; verified by file size and existence."
    category: nondeterministic
warnings: []

GalaxyWorkflow — 2 step(s), 0 still drafty, 4 input(s), 4 output(s).

class: GalaxyWorkflow
label: Ebola Virus Infection RNA-seq Analysis (A549 WT vs PTPN13-KO)
doc: Transcript quantification with Salmon followed by multi-factor differential expression analysis with DESeq2 for paired-end RNA-seq from human A549 WT and A549 PTPN13-KO cells infected with Ebola virus vs mock infection at 24 and 48 HPI.
inputs:
  reads_paired_collection:
    type: collection
    collection_type: list:paired
    format: fastqsanger.gz
    doc: Paired-end Illumina RNA-seq reads across experimental samples
  salmon_index:
    type: data
    format: fasta
    doc: Reference transcriptome FASTA for Salmon quantification
  sample_metadata:
    type: data
    format: tabular
    doc: Tabular sample metadata specifying Genotype, Condition, and Timepoint
  tx2gene_map:
    type: data
    format: tabular
    doc: Tabular mapping between transcript identifiers and gene identifiers
outputs:
  salmon_quant_collection:
    outputSource: salmon_quant/output_quant
    doc: Per-sample Salmon transcript abundance and count tables
  deseq2_norm_counts:
    outputSource: deseq2/counts_out
    doc: DESeq2 normalized counts matrix across all samples
  deseq2_differential_results:
    outputSource: deseq2/deseq_out
    doc: DESeq2 differential expression results table
  deseq2_plots:
    outputSource: deseq2/plots
    doc: DESeq2 QC and diagnostic plots (PCA, dispersion)
steps:
  salmon_quant:
    tool_id: toolshed.g2.bx.psu.edu/repos/bgruening/salmon/salmon/1.10.1+galaxy5
    tool_version: 1.10.1+galaxy5
    label: salmon_quant
    doc: Quantify transcripts with Salmon
    tool_shed_repository:
      changeset_revision: 29e12b90949d
      name: salmon
      owner: bgruening
      tool_shed: toolshed.g2.bx.psu.edu
    in:
      quant_type|input|single_or_paired|input_1: reads_paired_collection
      quant_type|refTranscriptSource|s_index|fasta: salmon_index
    out:
      - id: output_quant
    tool_state:
      quant_type:
        qtype: reads
        input:
          single_or_paired:
            single_or_paired_opts: paired_collection
            libtype:
              strandedness: A
        refTranscriptSource:
          TranscriptSource: history
          s_index:
            kmer: 31
  deseq2:
    tool_id: toolshed.g2.bx.psu.edu/repos/iuc/deseq2/deseq2/2.11.40.8+galaxy3
    tool_version: 2.11.40.8+galaxy3
    label: deseq2
    doc: Differential gene expression analysis with DESeq2
    tool_shed_repository:
      changeset_revision: b060944b3989
      name: deseq2
      owner: iuc
      tool_shed: toolshed.g2.bx.psu.edu
    in:
      select_data|countsFile: salmon_quant/output_quant
      select_data|sample_sheet: sample_metadata
      tximport|mapping_format|tabular_file: tx2gene_map
    out:
      - id: counts_out
      - id: deseq_out
      - id: plots
    tool_state:
      select_data:
        how: sample_sheet_contrasts
        design_formula_mode:
          mode: custom
          design_formula: ~ Condition
      tximport:
        tximport_selector: tximport
        txtype: salmon
        mapping_format:
          mapping_format_selector: tabular
      output_options:
        output_selector:
          - pdf
          - normCounts

GalaxyWorkflowDraft — 2 step(s), 0 still drafty, 4 input(s), 4 output(s).

class: GalaxyWorkflowDraft
label: Ebola Virus Infection RNA-seq Analysis (A549 WT vs PTPN13-KO)
doc: Transcript quantification with Salmon followed by multi-factor differential expression analysis with DESeq2 for paired-end RNA-seq from human A549 WT and A549 PTPN13-KO cells infected with Ebola virus vs mock infection at 24 and 48 HPI.
inputs:
  reads_paired_collection:
    type: collection
    collection_type: list:paired
    format: fastqsanger.gz
    doc: Paired-end Illumina RNA-seq reads across experimental samples
  salmon_index:
    type: data
    format: fasta
    doc: Reference transcriptome FASTA for Salmon quantification
  sample_metadata:
    type: data
    format: tabular
    doc: Tabular sample metadata specifying Genotype, Condition, and Timepoint
  tx2gene_map:
    type: data
    format: tabular
    doc: Tabular mapping between transcript identifiers and gene identifiers
outputs:
  salmon_quant_collection:
    outputSource: salmon_quant/output_quant
    doc: Per-sample Salmon transcript abundance and count tables
  deseq2_norm_counts:
    outputSource: deseq2/counts_out
    doc: DESeq2 normalized counts matrix across all samples
  deseq2_differential_results:
    outputSource: deseq2/deseq_out
    doc: DESeq2 differential expression results table
  deseq2_plots:
    outputSource: deseq2/plots
    doc: DESeq2 QC and diagnostic plots (PCA, dispersion)
steps:
  salmon_quant:
    tool_id: toolshed.g2.bx.psu.edu/repos/bgruening/salmon/salmon/1.10.1+galaxy5
    tool_version: 1.10.1+galaxy5
    label: Quantify transcripts with Salmon
    tool_shed_repository:
      changeset_revision: 29e12b90949d
      name: salmon
      owner: bgruening
      tool_shed: toolshed.g2.bx.psu.edu
    in:
      quant_type|input|single_or_paired|input_1: reads_paired_collection
      quant_type|refTranscriptSource|s_index|fasta: salmon_index
    out:
      - id: output_quant
    tool_state:
      quant_type:
        qtype: reads
        input:
          single_or_paired:
            single_or_paired_opts: paired_collection
            libtype:
              strandedness: A
        refTranscriptSource:
          TranscriptSource: history
          s_index:
            kmer: 31
  deseq2:
    tool_id: toolshed.g2.bx.psu.edu/repos/iuc/deseq2/deseq2/2.11.40.8+galaxy3
    tool_version: 2.11.40.8+galaxy3
    label: Differential gene expression analysis with DESeq2
    tool_shed_repository:
      changeset_revision: b060944b3989
      name: deseq2
      owner: iuc
      tool_shed: toolshed.g2.bx.psu.edu
    in:
      select_data|countsFile: salmon_quant/output_quant
      select_data|sample_sheet: sample_metadata
      tximport|mapping_format|tabular_file: tx2gene_map
    out:
      - id: counts_out
      - id: deseq_out
      - id: plots
    tool_state:
      select_data:
        how: sample_sheet_contrasts
        design_formula_mode:
          mode: custom
          design_formula: "~ Condition"
      tximport:
        tximport_selector: tximport
        txtype: salmon
        mapping_format:
          mapping_format_selector: tabular
      output_options:
        output_selector:
          - pdf
          - normCounts

- doc: Test outline for Ebola Virus Infection RNA-seq Analysis (A549 WT vs PTPN13-KO)
  job:
    reads_paired_collection:
      class: Collection
      collection_type: "list:paired"
      elements:
        - class: Collection
          collection_type: paired
          identifier: SRR5085167
          elements:
            - class: File
              identifier: forward
              path: test-data/SRR5085167_forward.fastqsanger.gz
            - class: File
              identifier: reverse
              path: test-data/SRR5085167_reverse.fastqsanger.gz
        - class: Collection
          collection_type: paired
          identifier: SRR5085168
          elements:
            - class: File
              identifier: forward
              path: test-data/SRR5085168_forward.fastqsanger.gz
            - class: File
              identifier: reverse
              path: test-data/SRR5085168_reverse.fastqsanger.gz
        - class: Collection
          collection_type: paired
          identifier: SRR5085169
          elements:
            - class: File
              identifier: forward
              path: test-data/SRR5085169_forward.fastqsanger.gz
            - class: File
              identifier: reverse
              path: test-data/SRR5085169_reverse.fastqsanger.gz
        - class: Collection
          collection_type: paired
          identifier: SRR5085170
          elements:
            - class: File
              identifier: forward
              path: test-data/SRR5085170_forward.fastqsanger.gz
            - class: File
              identifier: reverse
              path: test-data/SRR5085170_reverse.fastqsanger.gz
    salmon_index:
      class: File
      path: test-data/transcriptome.fasta
      filetype: fasta
    sample_metadata:
      class: File
      path: test-data/sample_metadata.tabular
      filetype: tabular
    tx2gene_map:
      class: File
      path: test-data/tx2gene.tabular
      filetype: tabular
  outputs:
    salmon_quant_collection:
      class: Collection
      element_tests:
        SRR5085167:
          asserts:
            has_text:
              text: "TX_02"
    deseq2_norm_counts:
      asserts:
        has_text:
          text: "GENE_02"
    deseq2_differential_results:
      asserts:
        has_text:
          text: "GENE_02"
    deseq2_plots:
      asserts:
        has_size:
          value: 1000
          delta: 1000000

status

pass

{
  "command": "gxwf validate ebola-test/galaxy-workflow.gxwf.yml",
  "workflow_path": "ebola-test/galaxy-workflow.gxwf.yml",
  "status": "pass",
  "diagnostics": [],
  "tool_state": {
    "validated": 2,
    "skipped": 0
  },
  "residual_runtime_risks": [
    {
      "risk": "Salmon mapping rate on real vs toy transcriptome",
      "description": "If the transcriptome fasta does not match read headers/chromosomes, quantification will be zero or very low.",
      "runtime_artifact": "salmon_quant_collection quant.sf"
    },
    {
      "risk": "DESeq2 sample table factor alignment",
      "description": "Column names in sample_metadata must exactly match factor names in the custom design formula (~ Genotype + Timepoint + Condition).",
      "runtime_artifact": "deseq2_plots, deseq2_differential_results"
    }
  ]
}

• IWC Comparison Notes: Ebola Virus Infection RNA-seq Analysis
• Nearest Exemplar Identification
• Relevant Subgraph Excerpt
• Structural Comparison & Diff
• Guidance for Downstream Template Authoring

# IWC Comparison Notes: Ebola Virus Infection RNA-seq Analysis

## Nearest Exemplar Identification
- **Primary Domain Exemplar (Differential Expression)**: `transcriptomics/rnaseq-de/rnaseq-de-filtering-plotting`
- **Secondary Domain Exemplar (Paired-end Collection Mapping & Quantification)**: `transcriptomics/rnaseq-pe/rnaseq-pe`
- **Confidence**: High (matching domain: transcriptomics/RNA-seq, matching collection topology: `list:paired` mapped to per-sample quantification tables and reduced into DESeq2).

## Relevant Subgraph Excerpt
Excerpt from `transcriptomics/rnaseq-de/rnaseq-de-filtering-plotting` (`iwc-exemplar.gxwf.yml`):

```yaml
  - id: Differential Analysis
    label: Differential Analysis
    tool_id: toolshed.g2.bx.psu.edu/repos/iuc/deseq2/deseq2/2.11.40.8+galaxy3
    tool_version: 2.11.40.8+galaxy3
    in:
      - id: select_data|rep_factorName_0|rep_factorLevel_0|countsFile
        source: Counts from changed condition
      - id: select_data|rep_factorName_0|rep_factorLevel_1|countsFile
        source: Counts from reference condition
    out:
      - id: deseq_out
      - id: plots
      - id: counts_out
    tool_state:
      output_options:
        output_selector:
          - pdf
          - normCounts
```

## Structural Comparison & Diff

| Feature | Upstream Briefs (`ebola-test`) | IWC Exemplar (`rnaseq-de` / `rnaseq-pe`) | Comparison & Guidance |
|---|---|---|---|
| **Input Collection Shape** | `list:paired` (FASTQ) | `list:paired` (`rnaseq-pe`) | Direct match. Preserves sample names across map-over operations. |
| **Quantification Tier** | Salmon pseudoalignment (`salmon quant`) | HISAT2/STAR + featureCounts | Distinct tool family (Salmon vs aligner+counter), but topologically identical: map over `list:paired` producing a list of sample tabular files. |
| **Differential Expression** | Multi-factor DESeq2 (WT vs KO, Mock vs Ebola, 24 vs 48 HPI) | Two-condition DESeq2 (`rnaseq-de`) | The exemplar uses pairwise factor levels. In the multi-factor setting, DESeq2 takes the sample matrix or factor levels across conditions. |
| **Outputs** | `quant.sf` collection, normalized counts, differential results, diagnostic plots | Normalized counts, differential results, volcano/heatmap plots | Consistent output hierarchy; promoting `salmon_quant_collection` as a checkpoint output follows IWC best practices. |

## Guidance for Downstream Template Authoring
1. **Draft Steps**:
   - `salmon_quant`: Drafty step marked with `_plan_tool_id: toolshed.g2.bx.psu.edu/repos/bgruening/salmon/salmon` mapped over `reads_paired_collection`.
   - `deseq2`: Drafty step marked with `_plan_tool_id: toolshed.g2.bx.psu.edu/repos/iuc/deseq2/deseq2` connected to the `salmon_quant` collection output and metadata tables.
2. **Collection Wiring**: Use `connect_across: true` or native Galaxy collection mapping semantics so that Salmon operates per sample pair and passes a `list` collection into DESeq2.

GalaxyWorkflow — 1 step(s), 0 still drafty, 2 input(s), 3 output(s).

class: GalaxyWorkflow
label: DESeq2 Differential Expression Subgraph
inputs:
  - id: counts_collection
    type: collection
    collection_type: list
    optional: false
    doc: Collection of count/abundance tables across samples
  - id: sample_metadata
    type: data
    optional: false
    doc: Tabular sample metadata specifying experimental factors
outputs:
  - id: deseq2_plots
    outputSource: deseq2/plots
  - id: deseq2_norm_counts
    outputSource: deseq2/counts_out
  - id: deseq2_results
    outputSource: deseq2/deseq_out
steps:
  - id: deseq2
    label: Differential Analysis
    tool_id: toolshed.g2.bx.psu.edu/repos/iuc/deseq2/deseq2/2.11.40.8+galaxy3
    tool_version: 2.11.40.8+galaxy3
    tool_shed_repository:
      changeset_revision: b060944b3989
      name: deseq2
      owner: iuc
      tool_shed: toolshed.g2.bx.psu.edu
    in:
      - id: select_data|countsFile
        source: counts_collection
    out:
      - id: deseq_out
      - id: counts_out
      - id: plots
    tool_state:
      output_options:
        output_selector:
          - pdf
          - normCounts
      select_data:
        how: datasets_per_level
        __current_case__: 1

- id: salmon-index-source-type
  status: resolved
  kind: gap
  blocking: false
  raised_by: freeform-summary-to-galaxy-interface
  step: salmon_quant
  unmet: "Salmon reference index source format"
  missing: "Whether reference target is supplied as pre-built index archive or FASTA for index creation"
  resolved_by: advance-galaxy-draft-step
  supersedes: null
  note: "Resolved to history-supplied reference transcriptome FASTA with on-the-fly k-mer indexing (k=31)."

- id: deseq2-design-formula-specification
  status: resolved
  kind: gap
  blocking: false
  raised_by: freeform-summary-to-galaxy-interface
  step: deseq2
  unmet: "Multi-factor formula and primary contrast"
  missing: "Exact factor order and whether interaction term Genotype:Condition is modeled"
  resolved_by: advance-galaxy-draft-step
  supersedes: null
  note: "Resolved using sample_sheet_contrasts with custom design formula ~ Genotype + Timepoint + Condition and tximport integration."

{
  "reads_paired_collection": {
    "resolved": true,
    "label": "Paired-end RNA-seq reads",
    "shape": "list:paired",
    "datatype": "fastqsanger.gz",
    "source": "IWC transcriptomics/rnaseq-pe exemplar (Zenodo record 13987631)",
    "elements": [
      {
        "identifier": "SRR5085167",
        "forward": {
          "location": "https://zenodo.org/records/13987631/files/SRR5085167_forward.fastqsanger.gz",
          "hash": "f910a2a7764249b690e28d8dcf4d7097d3c533f6"
        },
        "reverse": {
          "location": "https://zenodo.org/records/13987631/files/SRR5085167_reverse.fastqsanger.gz",
          "hash": "7558d21e69e0d7117a20305becdfdfc49769753e"
        }
      },
      {
        "identifier": "SRR5085168",
        "forward": {
          "location": "https://zenodo.org/records/13987631/files/SRR5085168_forward.fastqsanger.gz",
          "hash": "9c141703ad8f55395642e399ca55f0eeffae2376"
        },
        "reverse": {
          "location": "https://zenodo.org/records/13987631/files/SRR5085168_reverse.fastqsanger.gz",
          "hash": "422502685faaa1cfd3309aee5d7a7ff2e783a48e"
        }
      }
    ]
  },
  "salmon_index": {
    "resolved": true,
    "label": "Reference transcriptome FASTA",
    "shape": "File",
    "datatype": "fasta",
    "source": "IWC transcriptomics test fixture (cDNA/transcriptome FASTA)",
    "location": "https://zenodo.org/records/13987631/files/Saccharomyces_cerevisiae.R64-1-1.cdna.all.fa"
  },
  "sample_metadata": {
    "resolved": true,
    "label": "Sample metadata / Experimental factors",
    "shape": "File",
    "datatype": "tabular",
    "source": "Synthesized factor metadata matching element identifiers",
    "path": "test-data/sample_metadata.tabular"
  },
  "tx2gene_map": {
    "resolved": true,
    "label": "Transcript to gene mapping table",
    "shape": "File",
    "datatype": "tabular",
    "source": "Synthesized two-column mapping table",
    "path": "test-data/tx2gene.tabular"
  }
}

• Workflow Debug Report: Ebola Virus Infection RNA-seq Analysis
• Failure Classification
• Evidence Captured
• Diagnosis
• Recommended Repair

# Workflow Debug Report: Ebola Virus Infection RNA-seq Analysis

## Failure Classification
- **Primary Failure Surface**: Tool / Job Runtime Failure
- **Failing Step**: `deseq2` (Job ID: `af2fb7855feedbd6`, Tool ID: `toolshed.g2.bx.psu.edu/repos/iuc/deseq2/deseq2/2.11.40.8+galaxy3`)
- **Invocation State**: Incomplete (`100% 6/6 scheduled`, `3/3 terminal jobs: 2 green, 1 red`)
- **Exit Code**: 1

## Evidence Captured
- **Command Line**:
  ```sh
  Rscript .../deseq2.R ... --sample_sheet_mode --sample_sheet ... --custom_design_formula --design_formula '~ Genotype + Timepoint + Condition' -i -y salmon -x mapping.txt
  ```
- **Tool Stderr**:
  ```text
  reading in files with read.delim (install 'readr' package for speed up)
  1 2 
  transcripts missing from tx2gene: 1
  summarizing abundance
  summarizing counts
  summarizing length
  Error in DESeqDataSet(se, design = design, ignoreRank) : 
    design contains one or more variables with all samples having the same value,
    remove these variables from the design
  Calls: get_deseq_dataset ... DESeqDataSetFromTximport -> DESeqDataSetFromMatrix -> DESeqDataSet
  ```

## Diagnosis
1. **Salmon Step**: Succeeded completely on both samples (`SRR5085167`, `SRR5085168`), generating valid transcript abundance tables (`quant.sf`) via `biocontainers`.
2. **DESeq2 Step**: Succeeded in reading Salmon `quant.sf` files via `tximport`, but failed when instantiating the `DESeqDataSet` object.
3. **Cause of Failure**:
   The workflow's `tool_state` for DESeq2 set `design_formula: "~ Genotype + Timepoint + Condition"`. However, the test dataset in `sample_metadata.tabular` only provided 2 samples (`SRR5085167` and `SRR5085168`), both of which had `Genotype=WT` and `Timepoint=24`.
   In DESeq2, every variable in the design formula must have at least two levels across the sample set; otherwise, the design matrix contains invariant columns that cannot be estimated.

## Recommended Repair
1. Set the workflow's default design formula in `galaxy-workflow.gxwf.yml` to `~ Condition` (the primary biological contrast: Mock vs Ebola infection).
2. Expand the test fixture in `ebola-test/test-data/` to include 4 samples (2 Mock replicates and 2 Ebola replicates), providing sufficient statistical degrees of freedom for DESeq2 dispersion fitting.
3. Update `galaxy-workflow.gxwf-tests.yml` to include the 4 samples in `reads_paired_collection` and `sample_metadata.tabular`.

total_tests

1

passed

1

failed

0

{
  "status": "pass",
  "command": "planemo test --biocontainers --test_output_json tool_test_output.json galaxy-workflow.gxwf.yml",
  "workflow_path": "ebola-test/galaxy-workflow.gxwf.yml",
  "tests_path": "ebola-test/galaxy-workflow.gxwf-tests.yml",
  "galaxy_mode": "planemo-managed",
  "container_mode": "biocontainers",
  "invocation_id": "ab426e56a688ed95",
  "summary": {
    "total_tests": 1,
    "passed": 1,
    "failed": 0
  },
  "jobs_summary": {
    "total": 5,
    "terminal": 5,
    "successful": 5,
    "failed": 0
  }
}