This report was generated by the AREE workflows/rnaseq Nextflow scaffold.
+
+
+
+
+
+
Field
+
Value
+
+
+
+
+
workflow_name
+
aree-rnaseq
+
+
+
workflow_version
+
0.1.0-scaffold
+
+
+
mode
+
raw_reanalysis
+
+
+
study_id
+
TUCCI2025_HEAT_PRIMING
+
+
+
comparison_id
+
naive_hw_vs_naive_control
+
+
+
workflow_start
+
2026-10-13T16:54:07.700267-07:00
+
+
+
+
+
+
+
+
Warnings
+
+
+
No warnings recorded.
+
+
+
If mode is processed_results_harmonization, note that raw FASTQ QC (FastQC/fastp/Salmon mapping rate) could not be independently verified — see the warnings array above and docs/design.md section 7.
+
+
+
Sample exclusions and viral read QC
+
+
+
No samples were excluded from differential expression.
+
+
+
Viral read QC was not run (no --viral_reference).
+
+
+
+
+
Differential expression summary
+
+
+
Loaded 28984 features. 1606 features have padj < 0.05.
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
gene_id
+
baseMean
+
log2FoldChange
+
lfcSE
+
stat
+
pvalue
+
padj
+
+
+
+
+
132758537
+
50.81008
+
-4.2697482
+
0.3765238
+
-11.339917
+
0
+
0e+00
+
+
+
132755919
+
14.11383
+
22.1692463
+
2.9577342
+
7.495348
+
0
+
0e+00
+
+
+
132746458
+
118.98464
+
-1.2851644
+
0.1795983
+
-7.155773
+
0
+
0e+00
+
+
+
132739707
+
26.22425
+
-1.5771146
+
0.2271913
+
-6.941793
+
0
+
0e+00
+
+
+
132759335
+
33.82920
+
-1.7552263
+
0.2566270
+
-6.839600
+
0
+
0e+00
+
+
+
132735870
+
43.16419
+
-1.7914826
+
0.2660552
+
-6.733500
+
0
+
0e+00
+
+
+
132757131
+
14.92583
+
-2.2496023
+
0.3331864
+
-6.751784
+
0
+
0e+00
+
+
+
132758695
+
114.89966
+
-0.9798606
+
0.1454273
+
-6.737802
+
0
+
0e+00
+
+
+
132729316
+
354.73640
+
0.6547617
+
0.1004843
+
6.516057
+
0
+
2e-07
+
+
+
132728916
+
51.83823
+
-1.0374072
+
0.1600937
+
-6.480001
+
0
+
2e-07
+
+
+
+
+
+
+
+
Volcano plot
+
+
+
+
+
+
+
+
+
+
+
+
Provenance
+
Source file checksums, tool versions, and parameters are recorded in TUCCI2025_HEAT_PRIMING_naive_hw_vs_naive_control_manifest.json, emitted by the EMIT_MANIFEST process. This report does not duplicate that data verbatim; consult the manifest for the authoritative, machine-readable record.
+
+
Generated by the AREE workflows/rnaseq scaffold. This report template has not been rendered against real pipeline outputs in this build — see workflows/rnaseq/README.md.
This report was generated by the AREE workflows/rnaseq Nextflow scaffold.
+
+
+
+
+
+
Field
+
Value
+
+
+
+
+
workflow_name
+
aree-rnaseq
+
+
+
workflow_version
+
0.1.0-scaffold
+
+
+
mode
+
raw_reanalysis
+
+
+
study_id
+
TUCCI2025_HEAT_PRIMING
+
+
+
comparison_id
+
primed_hw_vs_naive_hw
+
+
+
workflow_start
+
2026-10-13T17:17:25.904826-07:00
+
+
+
+
+
+
+
+
Warnings
+
+
+
No warnings recorded.
+
+
+
If mode is processed_results_harmonization, note that raw FASTQ QC (FastQC/fastp/Salmon mapping rate) could not be independently verified — see the warnings array above and docs/design.md section 7.
+
+
+
Sample exclusions and viral read QC
+
+
+
No samples were excluded from differential expression.
+
+
+
Viral read QC was not run (no --viral_reference).
+
+
+
+
+
Differential expression summary
+
+
+
Loaded 28291 features. 24 features have padj < 0.05.
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
gene_id
+
baseMean
+
log2FoldChange
+
lfcSE
+
stat
+
pvalue
+
padj
+
+
+
+
+
132721657
+
39.19202
+
23.9871947
+
2.5630762
+
9.358752
+
0.00e+00
+
0.0000000
+
+
+
132755919
+
12.69731
+
-22.6397408
+
2.9577333
+
-7.654423
+
0.00e+00
+
0.0000000
+
+
+
132758537
+
18.12688
+
2.8246754
+
0.4524687
+
6.242808
+
0.00e+00
+
0.0000033
+
+
+
132726688
+
41.53752
+
3.7757002
+
0.6736429
+
5.604899
+
0.00e+00
+
0.0001187
+
+
+
132718184
+
123.95825
+
-0.8150614
+
0.1620407
+
-5.029979
+
5.00e-07
+
0.0022357
+
+
+
132731707
+
19.95903
+
3.4956171
+
0.7104960
+
4.919967
+
9.00e-07
+
0.0032875
+
+
+
132737063
+
361.89006
+
-0.5618985
+
0.1170663
+
-4.799832
+
1.60e-06
+
0.0051696
+
+
+
132736906
+
151.84663
+
-3.8452316
+
0.8201260
+
-4.688586
+
2.80e-06
+
0.0078362
+
+
+
132720481
+
22.32012
+
-3.8675302
+
0.8737802
+
-4.426205
+
9.60e-06
+
0.0214759
+
+
+
132730982
+
7.08161
+
-4.5564385
+
1.0318474
+
-4.415806
+
1.01e-05
+
0.0214759
+
+
+
+
+
+
+
+
Volcano plot
+
+
+
+
+
+
+
+
+
+
+
+
Provenance
+
Source file checksums, tool versions, and parameters are recorded in TUCCI2025_HEAT_PRIMING_primed_hw_vs_naive_hw_manifest.json, emitted by the EMIT_MANIFEST process. This report does not duplicate that data verbatim; consult the manifest for the authoritative, machine-readable record.
+
+
Generated by the AREE workflows/rnaseq scaffold. This report template has not been rendered against real pipeline outputs in this build — see workflows/rnaseq/README.md.
+
+
+
+
+
+
+
+
+
+
+
\ No newline at end of file
diff --git a/data/studies/TUCCI2025_HEAT_PRIMING/TUCCI2025_HEAT_PRIMING_primed_hw_vs_naive_hw_workflow_manifest.json b/data/studies/TUCCI2025_HEAT_PRIMING/TUCCI2025_HEAT_PRIMING_primed_hw_vs_naive_hw_workflow_manifest.json
new file mode 100644
index 0000000..597bd5c
--- /dev/null
+++ b/data/studies/TUCCI2025_HEAT_PRIMING/TUCCI2025_HEAT_PRIMING_primed_hw_vs_naive_hw_workflow_manifest.json
@@ -0,0 +1,122 @@
+{
+ "workflow_name": "aree-rnaseq",
+ "workflow_version": "0.1.0-scaffold",
+ "mode": "raw_reanalysis",
+ "study_id": "TUCCI2025_HEAT_PRIMING",
+ "comparison_id": "primed_hw_vs_naive_hw",
+ "input_files": [
+ {
+ "name": "TUCCI2025_HEAT_PRIMING_primed_hw_vs_naive_hw_dge_standardized.tsv",
+ "sha256": "f869028d8ae52f05cff1325f8942cb5b0bf2ba173f0154802770c20c714f2816"
+ }
+ ],
+ "parameters": {
+ "mode": "raw_reanalysis",
+ "study_id": "TUCCI2025_HEAT_PRIMING",
+ "comparison_id": "primed_hw_vs_naive_hw",
+ "outdir": "results/rnaseq/TUCCI2025_HEAT_PRIMING/primed_hw_vs_naive_hw",
+ "exclude_samples": [],
+ "control_levels": [
+ "Na\u00efve_Heatwave"
+ ],
+ "treatment_levels": [
+ "Primed_Heatwave"
+ ],
+ "replicate_unit": null,
+ "covariates": [],
+ "single_end": true,
+ "se_adapter": "AGATCGGAAGAGCACACGTCTGAACTCCAGTCA",
+ "tag_seq": true,
+ "viral_reference": null
+ },
+ "deseq2_design": {
+ "design": "~condition",
+ "covariates_requested": [],
+ "covariates_used": [],
+ "covariates_dropped_constant": [],
+ "tag_seq": true,
+ "length_model": "none: counts without length offsets (tag_seq)",
+ "n_libraries_or_units": 20,
+ "residual_df": 18
+ },
+ "software_versions": {
+ "DIFFERENTIAL_EXPRESSION_DESEQ2": {
+ "bioconductor-deseq2": "1.50.2",
+ "bioconductor-tximport": "1.38.2",
+ "r-base": "4.5.3"
+ },
+ "FASTQC": {
+ "fastqc": "0.12.1"
+ },
+ "SALMON_INDEX": {
+ "salmon": "2.8.0"
+ },
+ "SALMON_QUANT": {
+ "salmon": "2.8.0"
+ },
+ "SAMPLE_QC": {
+ "multiqc": "1.35"
+ },
+ "STANDARDIZE_OUTPUT": {
+ "pandas": "3.0.6",
+ "python": "3.11.17"
+ },
+ "TRIM_FASTP": {
+ "fastp": "1.4.0"
+ },
+ "EMIT_MANIFEST": {
+ "python": "3.11.17"
+ }
+ },
+ "software_versions_not_captured": {
+ "RENDER_REPORT": "renders this manifest, so it runs afterwards; its versions.yml is published to rnaseq/reports/"
+ },
+ "container_engine": null,
+ "reference": {
+ "genome_assembly": "GCF_026571515.1",
+ "annotation_version": "GCF_026571515.1-RS_2023_10",
+ "transcript_fasta": "data/reference/GCF_026571515.1/rna.fna",
+ "gtf": "data/reference/GCF_026571515.1/genomic.gtf",
+ "tx2gene": "data/reference/GCF_026571515.1/tx2gene.tsv",
+ "salmon_index": null,
+ "files": [
+ {
+ "name": "genomic.gtf",
+ "sha256": "99299a6252c91d941eee4ae579ff540ef08019317ae3c44a7927500cdd46eb0b",
+ "matches_bundle_provenance": true
+ },
+ {
+ "name": "reference_provenance.json",
+ "sha256": "a39261e783dccf164ed976d93a25abc1f871f0d47ffd437218041f3b6cfeb67b"
+ },
+ {
+ "name": "rna.fna",
+ "sha256": "2c45a6c9478b0ff312737772ee3c23019468042fd09bbf2a25a7f263cbfe4014",
+ "matches_bundle_provenance": true
+ },
+ {
+ "name": "tx2gene.tsv",
+ "sha256": "1f76e774cf67286927a3428f570f3f899246e7849e2a60a5a1459de2e396a961",
+ "matches_bundle_provenance": true
+ }
+ ],
+ "bundle": {
+ "bundle": "GCF_026571515.1",
+ "assembly_id": "ASM2657151v2",
+ "ncbi_assembly_accession": "GCF_026571515.1",
+ "annotation_source": "NCBI RefSeq GCF_026571515.1-RS_2023_10",
+ "annotation_release_matches_registry": true,
+ "date_generated": "2026-10-13",
+ "bundler_version": "1.0.0"
+ }
+ },
+ "workflow_start": "2026-10-13T17:17:25.904826-07:00",
+ "qc_metrics": {
+ "mean_reads_per_sample": null,
+ "mean_mapping_rate_percent": null,
+ "n_samples": null,
+ "note": "QC metrics are not populated in processed_results_harmonization mode or in this unexecuted scaffold run."
+ },
+ "viral_load": null,
+ "warnings": []
+}
\ No newline at end of file
diff --git a/data/studies/TUCCI2025_HEAT_PRIMING/ena_provenance.json b/data/studies/TUCCI2025_HEAT_PRIMING/ena_provenance.json
new file mode 100644
index 0000000..de27957
--- /dev/null
+++ b/data/studies/TUCCI2025_HEAT_PRIMING/ena_provenance.json
@@ -0,0 +1,43 @@
+{
+ "study_id": "TUCCI2025_HEAT_PRIMING",
+ "bioproject": "PRJNA1132140",
+ "date_generated": "2026-10-13",
+ "generated_by": "src/intake/ena_samplesheet.py",
+ "queries": {
+ "run_report": "https://www.ebi.ac.uk/ena/portal/api/filereport?accession=PRJNA1132140&result=read_run&fields=run_accession,sample_accession,sample_alias,experiment_accession,library_strategy,library_layout,library_selection,instrument_model,read_count,base_count,fastq_ftp,fastq_md5,fastq_bytes",
+ "sample_xml": "https://www.ebi.ac.uk/ena/browser/api/xml/"
+ },
+ "condition_attributes": [
+ "Treatment",
+ "Thermal stress"
+ ],
+ "n_runs": 40,
+ "n_samples": 40,
+ "library_strategies": [
+ "RNA-Seq"
+ ],
+ "library_layouts": [
+ "SINGLE"
+ ],
+ "group_sizes": {
+ "Na\u00efve_Heatwave": 10,
+ "Na\u00efve_No_Heatwave": 10,
+ "Primed_Heatwave": 10,
+ "Primed_No_Heatwave": 10
+ },
+ "min_group_size": 10,
+ "total_fastq_bytes": 13799357777,
+ "outputs": {
+ "data/studies/TUCCI2025_HEAT_PRIMING/samplesheet.tsv": "5345cc441b47ff78697a496bd125685e1af95a491fcedf1a9ac931084ca927ae",
+ "data/studies/TUCCI2025_HEAT_PRIMING/fastq_manifest.tsv": "a1e2c0c9c767294567f3402d4da8996282ed85ecfeee29c0413a38c8f7fe474c"
+ },
+ "library_strategy_filter": {
+ "kept": [
+ "RNA-Seq"
+ ],
+ "runs_by_strategy_before_filter": {
+ "AMPLICON": 40,
+ "RNA-Seq": 40
+ }
+ }
+}
diff --git a/data/studies/TUCCI2025_HEAT_PRIMING/fastq_manifest.tsv b/data/studies/TUCCI2025_HEAT_PRIMING/fastq_manifest.tsv
new file mode 100644
index 0000000..e084d85
--- /dev/null
+++ b/data/studies/TUCCI2025_HEAT_PRIMING/fastq_manifest.tsv
@@ -0,0 +1,41 @@
+run_accession mate url md5 bytes
+SRR29712776 1 ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR297/076/SRR29712776/SRR29712776.fastq.gz bf8c6cf2e4dbc42de31f9338b5d5ab83 224005838
+SRR29712777 1 ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR297/077/SRR29712777/SRR29712777.fastq.gz 9365501b38dc81665603c7d68945de55 334635612
+SRR29712778 1 ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR297/078/SRR29712778/SRR29712778.fastq.gz 349b3bcf3c020c3ee3c3758fe355383b 397331910
+SRR29712779 1 ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR297/079/SRR29712779/SRR29712779.fastq.gz b36e8c5734796f20dad5efc64b597821 404420577
+SRR29712780 1 ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR297/080/SRR29712780/SRR29712780.fastq.gz 557ec238ff9f49f34bb3683121832bf7 457624434
+SRR29712781 1 ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR297/081/SRR29712781/SRR29712781.fastq.gz 115f94087c94068adf9a2c91ccdd6900 392649391
+SRR29712782 1 ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR297/082/SRR29712782/SRR29712782.fastq.gz 2e1d624bb96945e44b014011bb15b309 326528631
+SRR29712783 1 ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR297/083/SRR29712783/SRR29712783.fastq.gz b79acb95070850fa0ac1f27195d70fbe 328432556
+SRR29712784 1 ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR297/084/SRR29712784/SRR29712784.fastq.gz 4ef681acd542cdfcbb41a1b79e281afd 344649215
+SRR29712785 1 ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR297/085/SRR29712785/SRR29712785.fastq.gz b4995d6de3251abfc13abe70d8f260ad 316102075
+SRR29712786 1 ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR297/086/SRR29712786/SRR29712786.fastq.gz d7cc66ec4f449e421542f67bf065b8ed 318083236
+SRR29712787 1 ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR297/087/SRR29712787/SRR29712787.fastq.gz 19c0e0af67e6f7f8326ed1c9969ee1f9 319668215
+SRR29712788 1 ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR297/088/SRR29712788/SRR29712788.fastq.gz 6bdac838a11fe7646bfa5bb30f0f2d57 330696910
+SRR29712789 1 ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR297/089/SRR29712789/SRR29712789.fastq.gz c72af95b8fd1dba1dccc38bdd121aad0 325755461
+SRR29712790 1 ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR297/090/SRR29712790/SRR29712790.fastq.gz 6cb8b4d8ac631e450ea92e80a4121fd1 318487239
+SRR29712791 1 ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR297/091/SRR29712791/SRR29712791.fastq.gz 6be421004eebb3551864a5c53a89a978 265508696
+SRR29712792 1 ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR297/092/SRR29712792/SRR29712792.fastq.gz 0adbb29c85ec2313fba130ba8965a793 525909036
+SRR29712793 1 ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR297/093/SRR29712793/SRR29712793.fastq.gz 10b7d69fa32dfeb2a01f0635ba1554e5 284644942
+SRR29712794 1 ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR297/094/SRR29712794/SRR29712794.fastq.gz 44770960994dabde7d40980609b15263 336605601
+SRR29712795 1 ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR297/095/SRR29712795/SRR29712795.fastq.gz ab473ca5224d92ce0c2ddf39aee3c649 356839630
+SRR29712796 1 ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR297/096/SRR29712796/SRR29712796.fastq.gz fd30d3a3bebe7e22e4cc19643a14511f 326642696
+SRR29712797 1 ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR297/097/SRR29712797/SRR29712797.fastq.gz f5501653ebdf68352eeeb082b7a9f11c 331773190
+SRR29712798 1 ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR297/098/SRR29712798/SRR29712798.fastq.gz 51dc3792eefd92d38f46f3573623d1dc 308551604
+SRR29712799 1 ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR297/099/SRR29712799/SRR29712799.fastq.gz d1bc554dc4592003bf5d4a5ded243382 315178651
+SRR29712800 1 ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR297/000/SRR29712800/SRR29712800.fastq.gz 28ac8ae8886da061a547c3007709c26c 321465991
+SRR29712801 1 ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR297/001/SRR29712801/SRR29712801.fastq.gz acbff4bf685b9097e46cd9778dc21d6d 311024770
+SRR29712802 1 ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR297/002/SRR29712802/SRR29712802.fastq.gz 9572c1a1c0c5c1d6fe4fe6a2eb878ffc 293369029
+SRR29712803 1 ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR297/003/SRR29712803/SRR29712803.fastq.gz 5ef710f9a5074b2aa6b97c7cc631f62e 415400920
+SRR29712804 1 ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR297/004/SRR29712804/SRR29712804.fastq.gz a337b3a3eb7afa7227d22d1901b7f862 306772397
+SRR29712805 1 ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR297/005/SRR29712805/SRR29712805.fastq.gz 1648d6bfc81b251f4cfe366e728df1bf 304136502
+SRR29712806 1 ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR297/006/SRR29712806/SRR29712806.fastq.gz 89a06647738d1e7d385b14174848e33c 306060557
+SRR29712807 1 ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR297/007/SRR29712807/SRR29712807.fastq.gz 5912389ddc9c0682f78d2b108cef3fea 302457006
+SRR29712808 1 ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR297/008/SRR29712808/SRR29712808.fastq.gz 63b58b18caa779c2a6f81c302c773c14 281394688
+SRR29712809 1 ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR297/009/SRR29712809/SRR29712809.fastq.gz eefdfe126ef9ca7bdd23d91beeee7d26 274447026
+SRR29712810 1 ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR297/010/SRR29712810/SRR29712810.fastq.gz eb682f12efcd687f458c2c7faf8a5443 299948505
+SRR29712811 1 ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR297/011/SRR29712811/SRR29712811.fastq.gz 466b4c847efdb13b86f8c244c9435c52 317176310
+SRR29712812 1 ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR297/012/SRR29712812/SRR29712812.fastq.gz 746c9d57a06d124ab245d5d83de6432e 324485146
+SRR29712813 1 ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR297/013/SRR29712813/SRR29712813.fastq.gz 59bb2fb003da6f84da152bcf914fc678 324201210
+SRR29712814 1 ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR297/014/SRR29712814/SRR29712814.fastq.gz 9eabbec5bdfef5d305ff02d0f8b86829 773263048
+SRR29712815 1 ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR297/015/SRR29712815/SRR29712815.fastq.gz 725d9164c2b65a3aa778c0cedbb2e2c0 453029326
diff --git a/data/studies/TUCCI2025_HEAT_PRIMING/replicate_dispersion_qc.tsv b/data/studies/TUCCI2025_HEAT_PRIMING/replicate_dispersion_qc.tsv
new file mode 100644
index 0000000..a255ca3
--- /dev/null
+++ b/data/studies/TUCCI2025_HEAT_PRIMING/replicate_dispersion_qc.tsv
@@ -0,0 +1,5 @@
+condition n_samples samples n_genes median_dispersion passes
+Naïve_Heatwave 10 Naive_HW_1,Naive_HW_10,Naive_HW_2,Naive_HW_3,Naive_HW_4,Naive_HW_5,Naive_HW_6,Naive_HW_7,Naive_HW_8,Naive_HW_9 845 0.08169 True
+Naïve_No_Heatwave 10 Naive_noHW_1,Naive_noHW_10,Naive_noHW_2,Naive_noHW_3,Naive_noHW_4,Naive_noHW_5,Naive_noHW_6,Naive_noHW_7,Naive_noHW_8,Naive_noHW_9 857 0.09977 True
+Primed_Heatwave 10 Primed_HW_1,Primed_HW_10,Primed_HW_2,Primed_HW_3,Primed_HW_4,Primed_HW_5,Primed_HW_6,Primed_HW_7,Primed_HW_8,Primed_HW_9 857 0.11639 True
+Primed_No_Heatwave 10 Primed_noHW_1,Primed_noHW_10,Primed_noHW_2,Primed_noHW_3,Primed_noHW_4,Primed_noHW_5,Primed_noHW_6,Primed_noHW_7,Primed_noHW_8,Primed_noHW_9 861 0.09169 True
diff --git a/data/studies/TUCCI2025_HEAT_PRIMING/samplesheet.tsv b/data/studies/TUCCI2025_HEAT_PRIMING/samplesheet.tsv
new file mode 100644
index 0000000..880680a
--- /dev/null
+++ b/data/studies/TUCCI2025_HEAT_PRIMING/samplesheet.tsv
@@ -0,0 +1,41 @@
+sample_id run_accession sample_accession sample_alias condition treatment thermal_stress clam_id library_layout instrument_model read_count
+Naive_HW_10 SRR29712776 SAMN42331788 Naive_HW_10 Naïve_Heatwave Naïve Heatwave Naive_HW_10 SINGLE Illumina NovaSeq 6000 7168739
+Naive_HW_9 SRR29712777 SAMN42331787 Naive_HW_9 Naïve_Heatwave Naïve Heatwave Naive_HW_9 SINGLE Illumina NovaSeq 6000 6960065
+Naive_HW_8 SRR29712778 SAMN42331786 Naive_HW_8 Naïve_Heatwave Naïve Heatwave Naive_HW_8 SINGLE Illumina NovaSeq 6000 8423412
+Naive_HW_7 SRR29712779 SAMN42331785 Naive_HW_7 Naïve_Heatwave Naïve Heatwave Naive_HW_7 SINGLE Illumina NovaSeq 6000 8574241
+Naive_HW_6 SRR29712780 SAMN42331784 Naive_HW_6 Naïve_Heatwave Naïve Heatwave Naive_HW_6 SINGLE Illumina NovaSeq 6000 9691130
+Naive_HW_5 SRR29712781 SAMN42331783 Naive_HW_5 Naïve_Heatwave Naïve Heatwave Naive_HW_5 SINGLE Illumina NovaSeq 6000 8244452
+Primed_noHW_10 SRR29712782 SAMN42331818 Primed_noHW_10 Primed_No_Heatwave Primed No Heatwave Primed_noHW_10 SINGLE Illumina NovaSeq 6000 6709087
+Primed_noHW_9 SRR29712783 SAMN42331817 Primed_noHW_9 Primed_No_Heatwave Primed No Heatwave Primed_noHW_9 SINGLE Illumina NovaSeq 6000 6822580
+Primed_noHW_8 SRR29712784 SAMN42331816 Primed_noHW_8 Primed_No_Heatwave Primed No Heatwave Primed_noHW_8 SINGLE Illumina NovaSeq 6000 7104321
+Primed_noHW_7 SRR29712785 SAMN42331815 Primed_noHW_7 Primed_No_Heatwave Primed No Heatwave Primed_noHW_7 SINGLE Illumina NovaSeq 6000 6571845
+Primed_noHW_6 SRR29712786 SAMN42331814 Primed_noHW_6 Primed_No_Heatwave Primed No Heatwave Primed_noHW_6 SINGLE Illumina NovaSeq 6000 6692604
+Primed_noHW_5 SRR29712787 SAMN42331813 Primed_noHW_5 Primed_No_Heatwave Primed No Heatwave Primed_noHW_5 SINGLE Illumina NovaSeq 6000 6741337
+Primed_noHW_4 SRR29712788 SAMN42331812 Primed_noHW_4 Primed_No_Heatwave Primed No Heatwave Primed_noHW_4 SINGLE Illumina NovaSeq 6000 6968334
+Primed_noHW_3 SRR29712789 SAMN42331811 Primed_noHW_3 Primed_No_Heatwave Primed No Heatwave Primed_noHW_3 SINGLE Illumina NovaSeq 6000 6757371
+Primed_noHW_2 SRR29712790 SAMN42331810 Primed_noHW_2 Primed_No_Heatwave Primed No Heatwave Primed_noHW_2 SINGLE Illumina NovaSeq 6000 6727295
+Primed_noHW_1 SRR29712791 SAMN42331809 Primed_noHW_1 Primed_No_Heatwave Primed No Heatwave Primed_noHW_1 SINGLE Illumina NovaSeq 6000 5486885
+Naive_HW_4 SRR29712792 SAMN42331782 Naive_HW_4 Naïve_Heatwave Naïve Heatwave Naive_HW_4 SINGLE Illumina NovaSeq 6000 11178944
+Naive_noHW_10 SRR29712793 SAMN42331808 Naive_noHW_10 Naïve_No_Heatwave Naïve No Heatwave Naive_noHW_10 SINGLE Illumina NovaSeq 6000 6181284
+Naive_noHW_9 SRR29712794 SAMN42331807 Naive_noHW_9 Naïve_No_Heatwave Naïve No Heatwave Naive_noHW_9 SINGLE Illumina NovaSeq 6000 7011997
+Naive_noHW_8 SRR29712795 SAMN42331806 Naive_noHW_8 Naïve_No_Heatwave Naïve No Heatwave Naive_noHW_8 SINGLE Illumina NovaSeq 6000 7412689
+Naive_noHW_7 SRR29712796 SAMN42331805 Naive_noHW_7 Naïve_No_Heatwave Naïve No Heatwave Naive_noHW_7 SINGLE Illumina NovaSeq 6000 6835926
+Naive_noHW_6 SRR29712797 SAMN42331804 Naive_noHW_6 Naïve_No_Heatwave Naïve No Heatwave Naive_noHW_6 SINGLE Illumina NovaSeq 6000 6924763
+Naive_noHW_5 SRR29712798 SAMN42331803 Naive_noHW_5 Naïve_No_Heatwave Naïve No Heatwave Naive_noHW_5 SINGLE Illumina NovaSeq 6000 6533480
+Naive_noHW_4 SRR29712799 SAMN42331802 Naive_noHW_4 Naïve_No_Heatwave Naïve No Heatwave Naive_noHW_4 SINGLE Illumina NovaSeq 6000 6611829
+Naive_noHW_3 SRR29712800 SAMN42331801 Naive_noHW_3 Naïve_No_Heatwave Naïve No Heatwave Naive_noHW_3 SINGLE Illumina NovaSeq 6000 6743976
+Naive_noHW_2 SRR29712801 SAMN42331800 Naive_noHW_2 Naïve_No_Heatwave Naïve No Heatwave Naive_noHW_2 SINGLE Illumina NovaSeq 6000 6512644
+Naive_noHW_1 SRR29712802 SAMN42331799 Naive_noHW_1 Naïve_No_Heatwave Naïve No Heatwave Naive_noHW_1 SINGLE Illumina NovaSeq 6000 6100702
+Naive_HW_3 SRR29712803 SAMN42331781 Naive_HW_3 Naïve_Heatwave Naïve Heatwave Naive_HW_3 SINGLE Illumina NovaSeq 6000 8793029
+Primed_HW_10 SRR29712804 SAMN42331798 Primed_HW_10 Primed_Heatwave Primed Heatwave Primed_HW_10 SINGLE Illumina NovaSeq 6000 6468236
+Primed_HW_9 SRR29712805 SAMN42331797 Primed_HW_9 Primed_Heatwave Primed Heatwave Primed_HW_9 SINGLE Illumina NovaSeq 6000 6348346
+Primed_HW_8 SRR29712806 SAMN42331796 Primed_HW_8 Primed_Heatwave Primed Heatwave Primed_HW_8 SINGLE Illumina NovaSeq 6000 6468392
+Primed_HW_7 SRR29712807 SAMN42331795 Primed_HW_7 Primed_Heatwave Primed Heatwave Primed_HW_7 SINGLE Illumina NovaSeq 6000 6356518
+Primed_HW_6 SRR29712808 SAMN42331794 Primed_HW_6 Primed_Heatwave Primed Heatwave Primed_HW_6 SINGLE Illumina NovaSeq 6000 5881714
+Primed_HW_5 SRR29712809 SAMN42331793 Primed_HW_5 Primed_Heatwave Primed Heatwave Primed_HW_5 SINGLE Illumina NovaSeq 6000 5750891
+Primed_HW_4 SRR29712810 SAMN42331792 Primed_HW_4 Primed_Heatwave Primed Heatwave Primed_HW_4 SINGLE Illumina NovaSeq 6000 6352796
+Primed_HW_3 SRR29712811 SAMN42331791 Primed_HW_3 Primed_Heatwave Primed Heatwave Primed_HW_3 SINGLE Illumina NovaSeq 6000 6570557
+Primed_HW_2 SRR29712812 SAMN42331790 Primed_HW_2 Primed_Heatwave Primed Heatwave Primed_HW_2 SINGLE Illumina NovaSeq 6000 6823321
+Primed_HW_1 SRR29712813 SAMN42331789 Primed_HW_1 Primed_Heatwave Primed Heatwave Primed_HW_1 SINGLE Illumina NovaSeq 6000 6815122
+Naive_HW_2 SRR29712814 SAMN42331780 Naive_HW_2 Naïve_Heatwave Naïve Heatwave Naive_HW_2 SINGLE Illumina NovaSeq 6000 16456702
+Naive_HW_1 SRR29712815 SAMN42331779 Naive_HW_1 Naïve_Heatwave Naïve Heatwave Naive_HW_1 SINGLE Illumina NovaSeq 6000 9737886
diff --git a/registry/studies/TUCCI2025_HEAT_PRIMING.yaml b/registry/studies/TUCCI2025_HEAT_PRIMING.yaml
new file mode 100644
index 0000000..5d19b0c
--- /dev/null
+++ b/registry/studies/TUCCI2025_HEAT_PRIMING.yaml
@@ -0,0 +1,222 @@
+# FIRST REAL CLAM STUDY REGISTERED IN AREE, and the first real study of a
+# species other than the Pacific oyster. Claimed 2026-10-12 from the WP5
+# backlog (docs/candidate_studies_mussels_clams.md, pool A: Manila clam heat).
+#
+# Registered from deposited metadata and the publication, which is CONFIRMED:
+# Tucci et al. 2025 (iScience, open access, PMC12314336) gives "BioProject ID:
+# PRJNA1132140" for both the RNA-seq and the 16S data, and its methods (2x2
+# factorial, 10 clams per group sequenced, 75 bp single-end) match the deposit.
+#
+# REANALYZED 2026-10-13 on Pori, fitted on clams with --tag_seq (no length
+# correction in Salmon or DESeq2). Heatwave vs control in naive clams: 1,606
+# of 28,984 genes at padj < 0.05. Primed vs naive under the heatwave: 24 of
+# 28,291. Both are upper bounds: tank, the unit of treatment, is not modelled
+# (limitations 2). Replicate dispersion QC passes (median 0.08-0.12 per
+# group). Salmon mapped 42-49% of reads; see limitations (1).
+
+study_id: TUCCI2025_HEAT_PRIMING
+simulated: false
+
+accessions:
+ doi: "10.1016/j.isci.2025.113108"
+ bioproject: "PRJNA1132140"
+ geo: null
+ sra: "SRR29712776-SRR29712815 (40 RNA-seq runs)"
+ ena: "PRJNA1132140"
+ proteomexchange: null
+ other: "BioSamples SAMN42331779-SAMN42331818 (one per clam, RNA-seq). The same BioProject holds 40 16S amplicon runs from the same RNA (BioSamples SAMN42638189-SAMN42638228), which are not used."
+
+citation: "Tucci CF, Milan M, Dalla Rovere G, Bernardini I, Ferraresso S, Franch R, Babbucci M, Monticelli G, Panin M, Patarnello T, Bargelloni L, Peruzza L (2025). Thermal priming mitigates the effects of lethal marine heatwaves on the Manila clam. iScience 28(8):113108. https://doi.org/10.1016/j.isci.2025.113108 (PMID 40755456)."
+
+species: "Ruditapes philippinarum"
+strain_or_population: "Commercial hatchery stock from SATMAR (France), adults 26.0 +/- 1.8 mm shell length and 3.75 +/- 0.81 g, purchased February 2022; experiments at the University of Padua marine station, Chioggia, in natural Venice lagoon seawater. Sex could not be determined (per the paper)."
+
+# AREE reanalyzes against the NCBI RefSeq annotation its Manila clam crosswalk
+# is built from. The authors mapped with STAR to the Xu et al. genome and
+# counted with featureCounts; that pipeline is not reproduced.
+genome_assembly: "ASM2657151v2"
+annotation_version: "GCF_026571515.1-RS_2023_10"
+
+assay_type: ["rnaseq"]
+analysis_mode: "raw_reanalysis"
+platform: "Illumina NovaSeq 6000, Lexogen QuantSeq 3' mRNA-seq (3' Tag) libraries prepared at CRIBI (University of Padua), 75 bp single-end (76 bp per ENA); 5.5-16.5 M reads per library (median 6.8 M); 13.8 GB FASTQ reported by ENA. Deposited as library_selection PolyA."
+
+data_availability:
+ raw_data_available: true
+ processed_data_available: false
+ status: "available"
+ access_notes: "Raw single-end FASTQ for all 40 RNA-seq libraries are public in ENA/SRA under PRJNA1132140 (first public 2025-06-28). The project also holds 40 paired-end 16S amplicon runs from the same RNA under the same Treatment and Thermal stress attributes; the sample sheet keeps only library_strategy RNA-Seq. Whether supplementary differential-expression tables exist was not checked; they would carry edgeR statistics after RUVSeq normalization and would not be reproduced here. Sample sheet and FASTQ manifest with ENA MD5s were generated from ENA metadata; sequence data are not committed."
+
+qc_status: "passed_with_warnings"
+analysis_status: "harmonized"
+
+# ---------------------------------------------------------------------------
+# DESIGN AS PUBLISHED. 2x2 factorial, priming (primed P / naive N) x marine
+# heatwave (HW H / control C). After 15 d acclimation (18 -> 25 C), primed
+# clams were held at 30 C for 7 d, then both groups rested 15 d at 25 C. Each
+# group was then split: a simulated 7 d heatwave oscillating daily 31-35 C,
+# or 25 C. All groups then recovered 15 d at 25 C. Survival was monitored
+# daily; RNA was taken at the end, day 46, from the digestive gland of 10
+# surviving clams per group. Each condition ran in duplicate 25 L aquaria with
+# 30 clams per tank. Measured outcome: about 25% mortality in naive clams
+# after the heatwave against about 4% in primed clams; none in either control.
+#
+# TWO COMPARISONS, PRESPECIFIED 2026-10-12, BEFORE ANY DATA WERE RUN.
+# naive_hw_vs_naive_control (NH vs NC) is the meta_analysis_primary
+# comparison: heatwave exposure in unprimed clams, the contrast that can
+# pool with other Manila clam heat studies (backlog pool A).
+# primed_hw_vs_naive_hw (PH vs NH) compares two groups that took the
+# same heatwave and differed in measured survival. It is classified
+# resilience: the closest thing in the mussel and clam backlog to a
+# tolerant-vs-sensitive contrast. See limitations (3) and (4).
+# Sample-sheet condition levels are Naïve_No_Heatwave, Naïve_Heatwave,
+# Primed_Heatwave, Primed_No_Heatwave (Treatment x Thermal stress).
+# ---------------------------------------------------------------------------
+comparisons:
+ - comparison_id: "naive_hw_vs_naive_control"
+ tissue: "digestive_gland"
+ life_stage: "adult"
+ stressor_original: "Simulated marine heatwave: 7 d with water temperature oscillating daily from 31 to 35 C (a 2015 Venice lagoon heatwave shifted +1 C), from a 25 C baseline, followed by 15 d recovery at 25 C (Tucci et al. 2025). BioSample attribute 'Thermal stress: Heatwave'."
+ stressor_standardized: "temperature"
+ exposure_intensity: "31-35 C daily oscillation vs constant 25 C"
+ exposure_duration: "7 d heatwave"
+ exposure_timing: "sampled on day 46, 15 d after the heatwave ended, at 25 C; survivors only"
+ control_condition: "Naive clams held at 25 C throughout (condition Naïve_No_Heatwave); 10 clams"
+ treatment_condition: "Naive clams exposed to the heatwave, sampled after 15 d recovery (condition Naïve_Heatwave); 10 surviving clams"
+ phenotype: "thermal_tolerance"
+ phenotype_direction: "not_applicable"
+ phenotype_unit: null
+ resilience_classification: "exposure_only"
+ sample_size: 20
+ biological_replicates: 2
+ results_file: "data/studies/TUCCI2025_HEAT_PRIMING/TUCCI2025_HEAT_PRIMING_naive_hw_vs_naive_control_dge_standardized.tsv"
+ meta_analysis_primary: true
+ - comparison_id: "primed_hw_vs_naive_hw"
+ tissue: "digestive_gland"
+ life_stage: "adult"
+ stressor_original: "Thermal priming: 7 d at 30 C (sublethal), 15 d rest at 25 C, then the simulated marine heatwave (7 d, 31-35 C daily) and 15 d recovery at 25 C. Both arms took the same heatwave; they differ only in priming. BioSample attributes 'Treatment: Primed' vs 'Treatment: Naïve', both 'Thermal stress: Heatwave'."
+ stressor_standardized: "temperature"
+ exposure_intensity: "priming 30 C for 7 d; heatwave 31-35 C daily oscillation in both arms"
+ exposure_duration: "7 d priming, then 7 d heatwave"
+ exposure_timing: "sampled on day 46, 15 d after the heatwave ended, at 25 C; survivors only"
+ control_condition: "Naive clams that took the heatwave (condition Naïve_Heatwave); about 25% of naive clams died after the heatwave; 10 survivors sequenced"
+ treatment_condition: "Primed clams that took the heatwave (condition Primed_Heatwave); about 4% died; 10 survivors sequenced"
+ phenotype: "thermal_tolerance"
+ phenotype_direction: "increase"
+ phenotype_unit: "percent_survival_after_heatwave (group level)"
+ resilience_classification: "resilience"
+ sample_size: 20
+ biological_replicates: 2
+ results_file: "data/studies/TUCCI2025_HEAT_PRIMING/TUCCI2025_HEAT_PRIMING_primed_hw_vs_naive_hw_dge_standardized.tsv"
+
+quality_flags:
+ - raw_data_available
+ - tank_level_replication
+ - single_timepoint
+
+limitations: >
+ (1) 3' Tag-Seq libraries (Lexogen QuantSeq, 75 bp single-end). Reads come
+ from the 3' end of each transcript, so counts do not scale with transcript
+ length, and the standard path's length correction (Salmon's effective-length
+ model, tximport length offsets in DESeq2) would be wrong: on simulated 3'
+ reads it called 26 of 40 isoform-switch genes differentially expressed with
+ no change in 3' reads. The run therefore sets tag_seq = true (fastp poly-X
+ trimming, Salmon --noLengthCorrection, DESeq2 on counts without length
+ offsets); the workflow manifest's deseq2_design.length_model must read
+ "none" for this study. Never run it without tag_seq. Gene-level 3' counts
+ also depend on 3' UTR annotation: genes whose RefSeq 3' end is wrong or
+ missing on this contig-level assembly will be under-counted. Salmon mapped
+ only 42-49% of reads (mean 46%) to the RefSeq transcriptome, consistent with
+ many 3' tags falling beyond annotated transcript ends; those genes are
+ measured on a fraction of their reads, or not at all. (2) Tank is the unit of
+ treatment: each condition ran in two 25 L tanks of 30 clams, so each arm
+ rests on 2 tanks. Which tank each sequenced clam came from is not in the
+ deposit or the paper, so the analysis cannot be fitted on tanks (as
+ LUTIER2022_OA_TIPPING is) unless the authors supply tank IDs. DECISION
+ (sr320, 2026-10-12): fit on clams now rather than wait for tank IDs. Each
+ arm's 10 clams are therefore treated as independent, so lfcSE ignores
+ tank-to-tank variation and is too small, and adjusted p-values are too
+ optimistic, by an amount that cannot be estimated from these data. The
+ study carries tank_level_replication and biological_replicates records 2;
+ treat its significance calls as an upper bound. If tank IDs arrive, refit
+ with --replicate_unit (two tanks per arm leaves 1 residual df per arm, so
+ expect far fewer calls) and record both.
+ (3) Survivor sampling. Naive clams lost about 25% to the heatwave and the
+ survivors were sampled 15 d later; primed clams lost about 4%. The naive
+ heatwave arm is therefore the more tolerant three-quarters of naive clams,
+ which biases primed_hw_vs_naive_hw toward the null and makes
+ naive_hw_vs_naive_control a recovery-phase contrast among survivors, not a
+ response at the peak of stress. (4) The resilience classification of
+ primed_hw_vs_naive_hw rests on a group-level survival difference. Priming
+ is an induced state, not a heritable line, and no sequenced clam was scored
+ for its own tolerance. The comparison says which genes differ between a
+ group that survives heatwaves better and one that does not, not which genes
+ make an individual tolerant. (5) Priming has a cost: primed clams had a
+ lower condition index with and without the heatwave, so primed vs naive
+ differences mix tolerance with that cost. (6) Single timepoint, single
+ tissue (digestive gland), one hatchery stock; sex not determined.
+ (7) Genome: RefSeq's only Manila clam annotation (ASM2657151v2) is
+ contig-level (N50 about 183 kb); gene-level evidence is unaffected, but see
+ (1) on 3' ends. (8) The independence of this study from PRJNA906528 (same
+ Padova group, 30-day 30 C exposure, digestive gland) must be confirmed
+ before the two are pooled as k = 2.
+
+provenance:
+ registered_by: sr320
+ date_registered: "2026-10-12"
+ curation_notes: >
+ Claimed with `aree start-study --accession PRJNA1132140 --study-id
+ TUCCI2025_HEAT_PRIMING`. Sample sheet and FASTQ manifest generated from
+ live ENA metadata with `aree fetch-samplesheet --bioproject PRJNA1132140
+ --study TUCCI2025_HEAT_PRIMING --condition-attribute Treatment
+ --condition-attribute "Thermal stress" --attribute Clam_ID
+ --library-strategy RNA-Seq`; the provenance records the filter (kept
+ RNA-Seq; 40 RNA-Seq and 40 AMPLICON runs before filtering). First
+ generated 2026-10-12 with PR #40's branch code, before --library-strategy
+ reached main; regenerated from main on 2026-10-13 after #40 merged. The
+ sample sheet and FASTQ manifest were byte-identical; only date_generated
+ in ena_provenance.json changed. TAG-SEQ: the run configuration sets
+ tag_seq = true (PR #24, merged into this branch 2026-10-12). LIBRARY-STRATEGY CHECK: unlike
+ LUTIER2022_OA_TIPPING, the AMPLICON label here is correct. The 40
+ AMPLICON runs are paired-end with their own BioSamples; the 40 RNA-Seq
+ runs are single-end, 76 bp, 5.5-16.5 M reads, matching the paper's 75 SE
+ 3' Tag libraries and its 16S V3-V4 250 bp paired-end amplicons.
+ REPLICATION CHECK: 40 distinct BioSamples and 40 distinct Clam_ID values
+ (Naive_noHW_1-10, Naive_HW_1-10, Primed_noHW_1-10, Primed_HW_1-10), 10
+ per group, matching the paper's 10 clams per group. Tank membership is
+ not recorded (limitation 2). Distinct names do not prove distinct
+ animals: run scripts/check_replicate_dispersion.py after quantification.
+ MAPPING: tissue 'digestive gland' (paper, RNAseq analyses) maps to
+ digestive_gland; 'Adult individuals' maps to adult. The reference bundle
+ for GCF_026571515.1 was fetched and verified with `aree fetch-reference`
+ on 2026-10-12: 65,336 transcripts, 47,388 genes, all of them in
+ rphil_gene_id_crosswalk.tsv, and the GTF names the registered release.
+ PUBLICATION: found by PubMed search for the design; the article's data
+ availability names PRJNA1132140. A follow-up from the same group (Martino
+ et al. 2026, ISME Commun, doi:10.1093/ismeco/ycag059) transplants primed
+ microbiomes; its data appear to be PRJNA1292094 (not checked) and it is a
+ separate experiment.
+ REANALYSIS (Pori, 2026-10-13): all 40 RNA-seq FASTQs (13.8 GB) were
+ downloaded from ENA and verified against ENA's MD5s; ENA throttled this
+ machine for most of a day, and 12 files needed fresh re-downloads over
+ HTTPS. Both comparisons ran natively (-profile local,workstation, 8 GB
+ resource config) with tag_seq = true, in one work directory: 126 tasks for
+ the primary comparison, then 4 for the second with 122 reused. Both
+ manifests record tag_seq true, length_model "none: counts without length
+ offsets (tag_seq)", 10 vs 10 clams, residual df 18, every reference file
+ matching the GCF_026571515.1 bundle provenance, and no warnings. Salmon
+ mapped 42.3-48.6% of reads per library. Replicate dispersion QC
+ (scripts/check_replicate_dispersion.py, replicate_dispersion_qc.tsv)
+ passes in all four groups: median dispersion 0.082 (Naïve_Heatwave),
+ 0.100 (Naïve_No_Heatwave), 0.116 (Primed_Heatwave), 0.092
+ (Primed_No_Heatwave), on about 850 genes per group with mean count >= 500
+ (few, because 3' tag libraries of ~7 M reads are shallow per gene).
+ Harmonized against rphil_gene_id_crosswalk.tsv: every gene resolves exact
+ (28,984 and 28,291 records). DESeq2 calls at padj < 0.05: 1,606 (632 up,
+ 974 down) for naive_hw_vs_naive_control, 24 (13 up, 11 down) for
+ primed_hw_vs_naive_hw; the latter is small, as limitations (3) predicts.
+ source_links:
+ - "https://www.ncbi.nlm.nih.gov/bioproject/PRJNA1132140"
+ - "https://www.ebi.ac.uk/ena/browser/view/PRJNA1132140"
+ - "https://doi.org/10.1016/j.isci.2025.113108"
+ - "https://pmc.ncbi.nlm.nih.gov/articles/PMC12314336/"
diff --git a/tests/test_tucci2025_study.py b/tests/test_tucci2025_study.py
new file mode 100644
index 0000000..59dbc93
--- /dev/null
+++ b/tests/test_tucci2025_study.py
@@ -0,0 +1,140 @@
+"""Curation tests for TUCCI2025_HEAT_PRIMING.
+
+The first real clam study: a 2x2 Manila clam priming x heatwave factorial,
+10 clams per group, 3' Tag-Seq of digestive gland. These tests pin the design
+read from ENA and the decisions that are easy to erode later: the 16S amplicon
+runs that share the BioProject are excluded, the two prespecified comparisons
+and their classifications, the tank and survivor caveats, and that the study is
+configuration runs the workflow's 3' Tag-seq path (tag_seq = true).
+"""
+from __future__ import annotations
+
+import csv
+import json
+import re
+from collections import Counter
+
+import pytest
+import yaml
+
+from common import DATA_DIR, REPO_ROOT, STUDIES_DIR
+
+STUDY_ID = "TUCCI2025_HEAT_PRIMING"
+STUDY_PATH = STUDIES_DIR / f"{STUDY_ID}.yaml"
+STUDY_DIR = DATA_DIR / "studies" / STUDY_ID
+CONFIG_PATH = REPO_ROOT / "config" / f"{STUDY_ID}.config"
+LEVELS = {"Naïve_No_Heatwave", "Naïve_Heatwave", "Primed_No_Heatwave", "Primed_Heatwave"}
+
+
+@pytest.fixture(scope="module")
+def study():
+ return yaml.safe_load(STUDY_PATH.read_text())
+
+
+@pytest.fixture(scope="module")
+def samplesheet():
+ with open(STUDY_DIR / "samplesheet.tsv", encoding="utf-8") as fh:
+ return list(csv.DictReader(fh, delimiter="\t"))
+
+
+@pytest.fixture(scope="module")
+def ena_provenance():
+ return json.loads((STUDY_DIR / "ena_provenance.json").read_text())
+
+
+def test_study_validates():
+ from intake.schema_validate import validate_study_file
+
+ result = validate_study_file(STUDY_PATH)
+ assert result.valid, result.errors
+
+
+def test_samplesheet_keeps_only_the_rnaseq_runs(samplesheet, ena_provenance):
+ """The BioProject also holds 40 paired-end 16S amplicon runs from the same RNA.
+ Counting them would double every group."""
+ assert ena_provenance["bioproject"] == "PRJNA1132140"
+ assert ena_provenance["library_strategy_filter"] == {
+ "kept": ["RNA-Seq"], "runs_by_strategy_before_filter": {"AMPLICON": 40, "RNA-Seq": 40}}
+ assert ena_provenance["library_layouts"] == ["SINGLE"]
+ assert len(samplesheet) == ena_provenance["n_runs"] == ena_provenance["n_samples"] == 40
+ assert {r["library_layout"] for r in samplesheet} == {"SINGLE"}
+
+
+def test_ten_distinct_clams_per_group(samplesheet):
+ assert Counter(r["condition"] for r in samplesheet) == dict.fromkeys(LEVELS, 10)
+ assert len({r["clam_id"] for r in samplesheet}) == 40
+ assert len({r["sample_accession"] for r in samplesheet}) == 40
+
+
+def test_two_prespecified_comparisons_with_one_primary(study):
+ comparisons = {c["comparison_id"]: c for c in study["comparisons"]}
+ assert set(comparisons) == {"naive_hw_vs_naive_control", "primed_hw_vs_naive_hw"}
+ primary = [cid for cid, c in comparisons.items() if c.get("meta_analysis_primary")]
+ assert primary == ["naive_hw_vs_naive_control"]
+
+
+def test_classifications_follow_what_was_measured(study):
+ """Heatwave vs control is an exposure among survivors. Primed vs naive under
+ the same heatwave compares groups with a measured survival difference."""
+ comparisons = {c["comparison_id"]: c for c in study["comparisons"]}
+ assert comparisons["naive_hw_vs_naive_control"]["resilience_classification"] == "exposure_only"
+ assert comparisons["primed_hw_vs_naive_hw"]["resilience_classification"] == "resilience"
+ for c in comparisons.values():
+ assert c["tissue"] == "digestive_gland"
+ assert c["stressor_standardized"] == "temperature"
+ assert c["phenotype"] == "thermal_tolerance"
+ assert "survivors only" in c["exposure_timing"]
+
+
+def test_tank_replication_and_tag_seq_are_declared(study):
+ assert "tank_level_replication" in study["quality_flags"]
+ assert all(c["biological_replicates"] == 2 for c in study["comparisons"])
+ assert "3' Tag" in study["platform"]
+ assert study["analysis_status"] == "harmonized"
+ assert study["qc_status"] == "passed_with_warnings"
+
+
+def test_config_levels_exist_in_the_samplesheet(samplesheet):
+ text = CONFIG_PATH.read_text(encoding="utf-8")
+ levels = set(re.findall(r"(?:control_level|treatment_level)\s*=\s*'([^']+)'", text))
+ assert levels == {"Naïve_No_Heatwave", "Naïve_Heatwave"}
+ assert levels <= {r["condition"] for r in samplesheet}
+ assert "single_end = true" in text
+ assert "GCF_026571515.1" in text
+
+
+def test_config_runs_the_tag_seq_path():
+ """QuantSeq 3' Tag-Seq: the standard path's length offsets would create false
+ fold changes from isoform switching. Never run this study without tag_seq."""
+ text = CONFIG_PATH.read_text(encoding="utf-8")
+ assert re.search(r"^\s*tag_seq\s*=\s*true\b", text, re.M)
+
+
+def test_command_line_comparison_id_is_not_shadowed():
+ """The workflow reads rnaseq.comparison_id before comparison_id; set inside the
+ rnaseq block it would silently name the second comparison after the first."""
+ text = CONFIG_PATH.read_text(encoding="utf-8")
+ block = re.search(r"rnaseq \{(.*?)\}", text, re.S).group(1)
+ assert re.search(r"comparison_id\s*=\s*null", block)
+ assert re.search(r"^\s*comparison_id\s*=\s*'naive_hw_vs_naive_control'", text, re.M)
+
+
+@pytest.mark.parametrize("cid", ["naive_hw_vs_naive_control", "primed_hw_vs_naive_hw"])
+def test_each_comparison_was_run_on_the_tag_seq_path(study, cid):
+ """The committed manifest must show the run that produced the results file:
+ tag_seq, no length offsets, 10 vs 10 clams, reference files as fetched."""
+ comp = {c["comparison_id"]: c for c in study["comparisons"]}[cid]
+ assert (REPO_ROOT / comp["results_file"]).exists()
+ manifest = json.loads((STUDY_DIR / f"{STUDY_ID}_{cid}_workflow_manifest.json").read_text())
+ assert manifest["parameters"]["tag_seq"] is True
+ assert manifest["deseq2_design"]["length_model"].startswith("none")
+ assert manifest["deseq2_design"]["n_libraries_or_units"] == 20
+ assert all(f.get("matches_bundle_provenance", True) for f in manifest["reference"]["files"])
+ assert manifest["warnings"] == []
+
+
+def test_replicate_dispersion_qc_passes():
+ with open(STUDY_DIR / "replicate_dispersion_qc.tsv", encoding="utf-8") as fh:
+ rows = list(csv.DictReader(fh, delimiter="\t"))
+ assert {r["condition"] for r in rows} == LEVELS
+ assert all(r["passes"] == "True" for r in rows)
