Interview-Driven Workflow Construction and Repair in Galaxy

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:

  Rscript .../deseq2.R ... --sample_sheet_mode --sample_sheet ... --custom_design_formula --design_formula '~ Genotype + Timepoint + Condition' -i -y salmon -x mapping.txt
  

• Tool Stderr:

  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.