[study] PERUZZA2023_HEATWAVE (PRJNA906528)

config/PERUZZA2023_HEATWAVE.config

@@ -0,0 +1,70 @@
+/*
+ * AREE :: run configuration for PERUZZA2023_HEATWAVE (PRJNA906528)
+ *
+ * The libraries are Lexogen QuantSeq 3' mRNA-Seq FWD (75 bp single-end; the
+ * paper's methods, section 2.10), so the run uses --tag_seq: fastp trims
+ * poly(A) tails, Salmon runs with --noLengthCorrection, and DESeq2 fits counts
+ * without length offsets. Never run this study without it. ENA labels the
+ * libraries library_selection PolyA, which hides this; the reads show it too
+ * (poly-A runs on the read, almost no poly-T).
+ *
+ * Sex is fitted as a covariate (~ sex + condition): the arms are unbalanced
+ * (20 C: 15 male, 5 female; 30 C: 10 and 10) and the authors found the
+ * heatwave response differs by sex. The deposited sex was predicted from
+ * digestive-gland transcriptomes by the authors' classifier, not observed;
+ * see limitations in registry/studies/PERUZZA2023_HEATWAVE.yaml.
+ *
+ * Tank is the unit of treatment (4 tanks per arm, 5 clams sampled per tank),
+ * but tank membership is not deposited, so replicate_unit cannot be set. The
+ * study carries tank_level_replication, as TUCCI2025_HEAT_PRIMING does.
+ *
+ *   nextflow run workflows/rnaseq -config config/PERUZZA2023_HEATWAVE.config \
+ *     -profile local,workstation
+ *
+ * The sample sheet and FASTQ manifest are generated, not hand-written:
+ *   aree fetch-samplesheet --bioproject PRJNA906528 --study PERUZZA2023_HEATWAVE \
+ *     --condition-attribute temp --attribute sex --library-strategy RNA-Seq
+ *
+ * The reference bundle comes from `aree fetch-reference --assembly ASM2657151v2`
+ * (docs/reference_bundles.md).
+ */
+
+includeConfig 'base.config'
+
+params {
+    mode       = 'raw_reanalysis'
+    single_end = true
+    tag_seq    = true   // QuantSeq 3' Tag-Seq: no length correction anywhere
+
+    rnaseq {
+        study_id           = 'PERUZZA2023_HEATWAVE'
+        // Null so a --comparison_id on the command line takes effect.
+        comparison_id      = null
+        genome_assembly    = 'GCF_026571515.1'
+        annotation_version = 'GCF_026571515.1-RS_2023_10'
+        processed_results  = null
+    }
+
+    // Prespecified primary comparison (meta_analysis_primary in the study YAML).
+    comparison_id = 'heatwave_30c_vs_control_20c'
+
+    sample_sheet = 'data/studies/PERUZZA2023_HEATWAVE/samplesheet.tsv'
+
+    // Condition levels are the BioSample attribute `temp`.
+    control_level   = '20'
+    treatment_level = '30'
+
+    covariates = 'sex'
+
+    // FASTQ are not committed (7.9 GB for the 40 RNA-seq runs). Verify
+    // against ENA's MD5s in data/studies/PERUZZA2023_HEATWAVE/fastq_manifest.tsv.
+    // Single-end: one file per run.
+    reads = 'data/raw/PERUZZA2023_HEATWAVE/SRR*.fastq.gz'
+
+    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
+
+    outdir = 'results/rnaseq/PERUZZA2023_HEATWAVE'
+}

data/studies/PERUZZA2023_HEATWAVE/PERUZZA2023_HEATWAVE_heatwave_30c_vs_control_20c_workflow_manifest.json

@@ -0,0 +1,127 @@
+{
+  "workflow_name": "aree-rnaseq",
+  "workflow_version": "0.1.0-scaffold",
+  "mode": "raw_reanalysis",
+  "study_id": "PERUZZA2023_HEATWAVE",
+  "comparison_id": "heatwave_30c_vs_control_20c",
+  "input_files": [
+    {
+      "name": "PERUZZA2023_HEATWAVE_heatwave_30c_vs_control_20c_dge_standardized.tsv",
+      "sha256": "cac70a4f116ca15fbd7d8b716817393f1f33370e1f89bd979292243ba4fd4923"
+    }
+  ],
+  "parameters": {
+    "mode": "raw_reanalysis",
+    "study_id": "PERUZZA2023_HEATWAVE",
+    "comparison_id": "heatwave_30c_vs_control_20c",
+    "outdir": "results/rnaseq/PERUZZA2023_HEATWAVE/heatwave_30c_vs_control_20c",
+    "exclude_samples": [],
+    "control_levels": [
+      "20"
+    ],
+    "treatment_levels": [
+      "30"
+    ],
+    "replicate_unit": null,
+    "covariates": [
+      "sex"
+    ],
+    "single_end": true,
+    "se_adapter": "AGATCGGAAGAGCACACGTCTGAACTCCAGTCA",
+    "tag_seq": true,
+    "viral_reference": null,
+    "exclude_genes": null
+  },
+  "deseq2_design": {
+    "design": "~sex + condition",
+    "covariates_requested": [
+      "sex"
+    ],
+    "covariates_used": [
+      "sex"
+    ],
+    "covariates_dropped_constant": [],
+    "tag_seq": true,
+    "length_model": "none: counts without length offsets (tag_seq)",
+    "exclude_genes": null,
+    "n_libraries_or_units": 40,
+    "residual_df": 37
+  },
+  "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_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": "/Users/sr320/aree-data/reference/GCF_026571515.1/salmon_index",
+    "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": "2026-10-17T00:01:30.595202-07:00",
+  "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": []
+}

data/studies/PERUZZA2023_HEATWAVE/ena_provenance.json

@@ -0,0 +1,44 @@
+{
+  "study_id": "PERUZZA2023_HEATWAVE",
+  "bioproject": "PRJNA906528",
+  "date_generated": "2026-10-15",
+  "generated_by": "src/intake/ena_samplesheet.py",
+  "queries": {
+    "run_report": "https://www.ebi.ac.uk/ena/portal/api/filereport?accession=PRJNA906528&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,submitted_ftp,submitted_md5,submitted_bytes,submitted_format",
+    "sample_xml": "https://www.ebi.ac.uk/ena/browser/api/xml/<sample_accessions>"
+  },
+  "condition_attributes": [
+    "temp"
+  ],
+  "n_runs": 40,
+  "n_samples": 40,
+  "library_strategies": [
+    "RNA-Seq"
+  ],
+  "library_layouts": [
+    "SINGLE"
+  ],
+  "group_sizes": {
+    "20": 20,
+    "30": 20
+  },
+  "min_group_size": 20,
+  "total_fastq_bytes": 7900304210,
+  "fastq_sources": {
+    "fastq_ftp": 40,
+    "submitted_ftp": 0
+  },
+  "outputs": {
+    "data/studies/PERUZZA2023_HEATWAVE/samplesheet.tsv": "0429ae867bd0009cca418e848e7e1c65c61b32e27cd68e4437ef7ea7f53d598b",
+    "data/studies/PERUZZA2023_HEATWAVE/fastq_manifest.tsv": "f39a9869be0bd86fe04e89b1d479d632e958942551109fc5fb14229c25f3868d"
+  },
+  "library_strategy_filter": {
+    "kept": [
+      "RNA-Seq"
+    ],
+    "runs_by_strategy_before_filter": {
+      "AMPLICON": 39,
+      "RNA-Seq": 40
+    }
+  }
+}

data/studies/PERUZZA2023_HEATWAVE/fastq_manifest.tsv

@@ -0,0 +1,41 @@
+run_accession	mate	url	md5	bytes	source
+SRR22444642	1	ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR224/042/SRR22444642/SRR22444642.fastq.gz	cf9352e5b70effd1a1f5ee96313b5858	197762464	fastq_ftp
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data/studies/PERUZZA2023_HEATWAVE/replicate_dispersion_qc.tsv

@@ -0,0 +1,3 @@
+condition	n_samples	samples	n_genes	median_dispersion	passes
+20	20	CTRL-107,CTRL-115,CTRL-122,CTRL-160,CTRL-167,CTRL-188,CTRL-190,CTRL-2,CTRL-212,CTRL-227,CTRL-233,CTRL-28,CTRL-38,CTRL-52,CTRL-65,CTRL-71,CTRL-77,CTRL-87,CTRL-94,CTRL-95	903	0.09657	True
+30	20	HW-113,HW-117,HW-145,HW-151,HW-154,HW-157,HW-159,HW-163,HW-165,HW-17,HW-19,HW-218,HW-238,HW-43,HW-46,HW-61,HW-85,HW-88,HW-91,HW-96	901	0.09313	True

data/studies/PERUZZA2023_HEATWAVE/samplesheet.tsv

@@ -0,0 +1,41 @@
+sample_id	run_accession	sample_accession	sample_alias	condition	temp	sex	library_layout	instrument_model	read_count
+CTRL-87	SRR22444642	SAMN31927501	CTRL-87	20	20	male	SINGLE	Illumina NovaSeq 6000	7046306
+CTRL-77	SRR22444643	SAMN31927500	CTRL-77	20	20	female	SINGLE	Illumina NovaSeq 6000	7807579
+CTRL-65	SRR22444644	SAMN31927499	CTRL-65	20	20	male	SINGLE	Illumina NovaSeq 6000	6418153
+CTRL-52	SRR22444645	SAMN31927498	CTRL-52	20	20	male	SINGLE	Illumina NovaSeq 6000	8193471
+CTRL-38	SRR22444646	SAMN31927497	CTRL-38	20	20	female	SINGLE	Illumina NovaSeq 6000	6495378
+CTRL-28	SRR22444647	SAMN31927496	CTRL-28	20	20	female	SINGLE	Illumina NovaSeq 6000	6740638
+HW-96	SRR22444648	SAMN31927531	HW-96	30	30	female	SINGLE	Illumina NovaSeq 6000	6450084
+HW-88	SRR22444649	SAMN31927530	HW-88	30	30	female	SINGLE	Illumina NovaSeq 6000	8473431
+HW-85	SRR22444650	SAMN31927529	HW-85	30	30	male	SINGLE	Illumina NovaSeq 6000	7650513
+HW-61	SRR22444651	SAMN31927528	HW-61	30	30	male	SINGLE	Illumina NovaSeq 6000	6585959
+HW-43	SRR22444652	SAMN31927527	HW-43	30	30	male	SINGLE	Illumina NovaSeq 6000	7837570
+HW-19	SRR22444653	SAMN31927526	HW-19	30	30	female	SINGLE	Illumina NovaSeq 6000	7935526
+HW-165	SRR22444654	SAMN31927525	HW-165	30	30	male	SINGLE	Illumina NovaSeq 6000	6675602
+HW-157	SRR22444655	SAMN31927524	HW-157	30	30	female	SINGLE	Illumina NovaSeq 6000	6338603
+HW-117	SRR22444656	SAMN31927523	HW-117	30	30	male	SINGLE	Illumina NovaSeq 6000	5670438
+HW-113	SRR22444657	SAMN31927522	HW-113	30	30	male	SINGLE	Illumina NovaSeq 6000	7384959
+CTRL-233	SRR22444658	SAMN31927495	CTRL-233	20	20	male	SINGLE	Illumina NovaSeq 6000	7960760
+CTRL-95	SRR22444659	SAMN31927521	CTRL-95	20	20	male	SINGLE	Illumina NovaSeq 6000	6504754
+CTRL-94	SRR22444660	SAMN31927520	CTRL-94	20	20	male	SINGLE	Illumina NovaSeq 6000	7520270
+CTRL-71	SRR22444661	SAMN31927519	CTRL-71	20	20	male	SINGLE	Illumina NovaSeq 6000	6379912
+CTRL-2	SRR22444662	SAMN31927518	CTRL-2	20	20	male	SINGLE	Illumina NovaSeq 6000	5953785
+CTRL-212	SRR22444663	SAMN31927517	CTRL-212	20	20	male	SINGLE	Illumina NovaSeq 6000	6781876
+CTRL-190	SRR22444664	SAMN31927516	CTRL-190	20	20	male	SINGLE	Illumina NovaSeq 6000	7251871
+CTRL-188	SRR22444665	SAMN31927515	CTRL-188	20	20	female	SINGLE	Illumina NovaSeq 6000	7481513
+CTRL-160	SRR22444666	SAMN31927514	CTRL-160	20	20	male	SINGLE	Illumina NovaSeq 6000	7601195
+CTRL-115	SRR22444667	SAMN31927513	CTRL-115	20	20	male	SINGLE	Illumina NovaSeq 6000	6988848
+CTRL-107	SRR22444668	SAMN31927512	CTRL-107	20	20	female	SINGLE	Illumina NovaSeq 6000	6891927
+CTRL-227	SRR22444669	SAMN31927494	CTRL-227	20	20	male	SINGLE	Illumina NovaSeq 6000	7466572
+HW-91	SRR22444670	SAMN31927511	HW-91	30	30	male	SINGLE	Illumina NovaSeq 6000	5084954
+HW-46	SRR22444671	SAMN31927510	HW-46	30	30	male	SINGLE	Illumina NovaSeq 6000	7265663
+HW-238	SRR22444672	SAMN31927509	HW-238	30	30	female	SINGLE	Illumina NovaSeq 6000	8343071
+HW-218	SRR22444673	SAMN31927508	HW-218	30	30	female	SINGLE	Illumina NovaSeq 6000	7974122
+HW-17	SRR22444674	SAMN31927507	HW-17	30	30	female	SINGLE	Illumina NovaSeq 6000	7527531
+HW-163	SRR22444675	SAMN31927506	HW-163	30	30	female	SINGLE	Illumina NovaSeq 6000	6736918
+HW-159	SRR22444676	SAMN31927505	HW-159	30	30	female	SINGLE	Illumina NovaSeq 6000	7994568
+HW-154	SRR22444677	SAMN31927504	HW-154	30	30	male	SINGLE	Illumina NovaSeq 6000	7714479
+HW-151	SRR22444678	SAMN31927503	HW-151	30	30	female	SINGLE	Illumina NovaSeq 6000	6267074
+HW-145	SRR22444679	SAMN31927502	HW-145	30	30	male	SINGLE	Illumina NovaSeq 6000	5876012
+CTRL-167	SRR22444680	SAMN31927493	CTRL-167	20	20	male	SINGLE	Illumina NovaSeq 6000	7520788
+CTRL-122	SRR22444681	SAMN31927492	CTRL-122	20	20	male	SINGLE	Illumina NovaSeq 6000	5557146