warning public, importable

id	wf_20260527_rnaflow-galaxy-native-rnaseq-reproduction
workflow_class	rnaseq_differential_expression
validation_profile	rnaseq_differential_expression
validation_status	warning
review_status	tested
source_type	github_repository
source_ref	f2d26a3418adc10d24b5042ad653081a9500245b
source_url	https://github.com/hoelzer-lab/rnaflow
galaxy_instance	https://usegalaxy.org
galaxy_history_id	bbd44e69cb8906b529dc497e1d783b61
execution_surface	galaxy-cli+galaxy-api-fallback
created	2026-05-27
updated	2026-06-13
tags	rnaseq, differential-expression, deseq2, hisat2, featurecounts, rnaflow, real-world

Entry README

RNAflow Galaxy-native RNA-seq reproduction

Summary

This entry reproduces the human RNA-seq test data from hoelzer-lab/rnaflow in Galaxy using the same core analysis stages: FastQC, HISAT2, featureCounts, and DESeq2. The public Galaxy history is the primary artifact.

Run Snapshot

Target: hoelzer-lab/rnaflow at source ref f2d26a3418adc10d24b5042ad653081a9500245b.

Public/importable Galaxy history: RNAflow real-world GitHub reproduction 2026-05-27.

Validation status: pass with warnings. The controlled website status is warning because the result is a useful Galaxy-native reproduction with documented caveats.

Source

Type: github_repository
URL: https://github.com/hoelzer-lab/rnaflow
Reference: f2d26a3418adc10d24b5042ad653081a9500245b
Data: test-data/hsa
Samples: four single-end FASTQ files, two MAQCA replicates and two MAQCB replicates

Galaxy Reproduction

History: https://usegalaxy.org/histories/view?id=bbd44e69cb8906b529dc497e1d783b61
Public: yes
Importable: yes
Execution surface: galaxy-cli+galaxy-api-fallback
Galaxy instance: https://usegalaxy.org
Galaxy history id: bbd44e69cb8906b529dc497e1d783b61

What Ran

Uploaded the RNAflow test-data/hsa FASTQs and DESeq2 sample sheet.
Ran FastQC on all four samples.
Ran HISAT2 against the built-in hg38 reference.
Ran featureCounts with the built-in hg38 annotation, single-end unstranded mode.
Ran DESeq2 for MAQCB compared with reference MAQCA.

Key Metrics

FASTQ samples: 4, each with 100000 reads.
HISAT2 overall alignment rate: 79.60% to 80.59%.
featureCounts assigned reads: 62228 to 62675.
DESeq2 raw rows: 28395.
DESeq2 finite rows after removing filtered NA statistics: 6744.
DESeq2 genes with adjusted p-value below 0.05: 979.

Validation

Status: warning
Profile: rnaseq_differential_expression

The status is warning rather than pass because this is a Galaxy-native reproduction of the RNAflow test workflow, not an exact Nextflow/conda execution of RNAflow itself. HISAT2 and DESeq2 were submitted through a narrow direct Galaxy API fallback during the run because the installed galaxy-cli did not yet normalize nested data input payloads for those wrappers.

Files

Metadata: metadata.yaml
Validation report: validation_report.json
Provenance: provenance.json

The original run also produced a run_summary.md; this page carries the reviewer-facing run summary while the validation report carries the machine-readable checks and metrics.

Entry id: wf_20260527_rnaflow-galaxy-native-rnaseq-reproduction

Validation Summary

Status	warning
Profile	rnaseq_differential_expression
Checks	13

Checks

Name	Status	Details
fresh_galaxy_history	pass	{"evidence": {"history_id": "bbd44e69cb8906b529dc497e1d783b61", "url": "https://usegalaxy.org/histories/view?id=bbd44e69cb8906b529dc497e1d783b61"}}
source_metadata	pass	{"evidence": {"conditions": {"MAQCA": 2, "MAQCB": 2}, "layout": "single-end", "samples": 4, "source_ref": "f2d26a3418adc10d24b5042ad653081a9500245b", "source_url": "https://github.com/hoelzer-lab/rnaflow", "strandedness": "unstranded"}}
all_galaxy_jobs_ok	pass	{"evidence": {"non_ok_job_count": 0, "ok_job_count": 18}}
fastq_integrity	pass	{"evidence": {"fastq_count": 4, "local_read_count_each": 100000}}
fastqc_basic_stats	pass	{"evidence": [{"filename": "SRR3670977_sub.fastq.gz", "gc_percent": 45, "sequence_length": "35-76", "total_sequences": 100000}, {"filename": "SRR3670978_sub.fastq.gz", "gc_percent": 45, "sequence_length": "35-76", "total_sequences": 100000}, {"filename": "SRR3670985_sub.fastq.gz", "gc_percent": 45, "sequence_length": "35-76", "total_sequences": 100000}, {"filename": "SRR3670986_sub.fastq.gz", "gc_percent": 45, "sequence_length": "35-76", "total_sequences": 100000}]}
hisat2_alignment	pass	{"evidence": [{"overall_alignment_rate_percent": 80.48, "reads": 100000, "sample": "SRR3670977_sub"}, {"overall_alignment_rate_percent": 80.59, "reads": 100000, "sample": "SRR3670978_sub"}, {"overall_alignment_rate_percent": 79.72, "reads": 100000, "sample": "SRR3670985_sub"}, {"overall_alignment_rate_percent": 79.6, "reads": 100000, "sample": "SRR3670986_sub"}]}
featurecounts_assigned_reads	pass	{"evidence": [{"assigned": 62322, "sample": "MAQCA_rep1", "unassigned_multimapping": 23985, "unassigned_unmapped": 19516}, {"assigned": 62228, "sample": "MAQCA_rep2", "unassigned_multimapping": 23825, "unassigned_unmapped": 19408}, {"assigned": 62675, "sample": "MAQCB_rep1", "unassigned_multimapping": 16290, "unassigned_unmapped": 20277}, {"assigned": 62618, "sample": "MAQCB_rep2", "unassigned_multimapping": 16513, "unassigned_unmapped": 20396}]}
deseq2_completed	pass	{"evidence": {"finite_rows": 6744, "raw_rows": 28395, "significant_padj_lt_0_05": 979}}
raw_de_table_contract	warning	{"evidence": {"passed": false, "reason": "Galaxy DESeq2 output is readable but lacks the exact header names expected by the local DE-table contract and includes NA p-values/padj for filtered genes."}}
finite_de_table_contract	pass	{"evidence": {"passed": true, "rows_checked": 6744}}
history_public_importable	pass	{"evidence": {"importable": true, "published": true, "verified_with": "unauthenticated Galaxy API"}}
execution_surface	warning	{"evidence": {"fallback": "direct Galaxy API for HISAT2 and DESeq2 nested dataset/collection payloads", "primary": "galaxy-cli", "reason": "The installed galaxy-cli used during the run did not normalize nested data fields such as library\|input_1 for HISAT2."}}
method_equivalence	warning	{"evidence": {"note": "Galaxy-native reproduction of RNAflow test data using matching HISAT2, featureCounts, and DESeq2 wrappers; not an exact Nextflow/conda RNAflow execution."}}

Warnings

Galaxy-native reproduction of RNAflow test data, not an exact Nextflow/conda RNAflow execution.
HISAT2 and DESeq2 used a narrow direct Galaxy API fallback because the installed galaxy-cli did not normalize nested dataset fields during this run.
The raw Galaxy DESeq2 table is readable but includes filtered NA statistics and needed a finite-row header-normalized copy for strict DE-table validation.

Provenance Summary

execution_surface	galaxy-cli+galaxy-api-fallback
created	2026-05-27
source_url	https://github.com/hoelzer-lab/rnaflow
source_ref	f2d26a3418adc10d24b5042ad653081a9500245b
galaxy_history_url	https://usegalaxy.org/histories/view?id=bbd44e69cb8906b529dc497e1d783b61

Galaxy Tools

Name	Tool ID
DESeq2	toolshed.g2.bx.psu.edu/repos/iuc/deseq2/deseq2/2.11.40.8+galaxy2
FastQC	toolshed.g2.bx.psu.edu/repos/devteam/fastqc/fastqc/0.74+galaxy1
HISAT2	toolshed.g2.bx.psu.edu/repos/iuc/hisat2/hisat2/2.2.2+galaxy0
featureCounts	toolshed.g2.bx.psu.edu/repos/iuc/featurecounts/featurecounts/2.1.1+galaxy0

Fallbacks

Direct Galaxy API submission for HISAT2 nested data input payloads.
Direct Galaxy API submission for DESeq2 nested collection/sample-sheet payloads.

Input Sources

Name	URL
RNAflow single-end sample table	https://raw.githubusercontent.com/hoelzer-lab/rnaflow/f2d26a3418adc10d24b5042ad653081a9500245b/test-data/hsa/input.se.hsa_small.csv
SRR3670977_sub.fastq.gz	https://raw.githubusercontent.com/hoelzer-lab/rnaflow/f2d26a3418adc10d24b5042ad653081a9500245b/test-data/hsa/SRR3670977_sub.fastq.gz
SRR3670978_sub.fastq.gz	https://raw.githubusercontent.com/hoelzer-lab/rnaflow/f2d26a3418adc10d24b5042ad653081a9500245b/test-data/hsa/SRR3670978_sub.fastq.gz
SRR3670985_sub.fastq.gz	https://raw.githubusercontent.com/hoelzer-lab/rnaflow/f2d26a3418adc10d24b5042ad653081a9500245b/test-data/hsa/SRR3670985_sub.fastq.gz
SRR3670986_sub.fastq.gz	https://raw.githubusercontent.com/hoelzer-lab/rnaflow/f2d26a3418adc10d24b5042ad653081a9500245b/test-data/hsa/SRR3670986_sub.fastq.gz