# RNA-seq: fastp trim -> salmon quant -> MultiQC -> DESeq2.
# Snakemake 8+. Run `snakemake -n` (dry run) to inspect the static DAG before executing.
# Reproducibility note: container tags are PINNED (never :latest). Prefer a @sha256: digest over a
# version tag for bit-reproducibility - a re-pushed version tag silently changes the tool and busts
# the rerun cache. Activate deployment with `snakemake --sdm apptainer --cores N`.

configfile: "config/config.yaml"

SAMPLES = config["samples"]
SALMON_INDEX = config["salmon_index"]

# Constrain the sample wildcard: greedy `.+` would otherwise swallow path separators and adjacent
# tokens, silently mis-routing a sample whose name contains an underscore or slash.
wildcard_constraints:
    sample = r"[A-Za-z0-9\-]+"

rule all:
    input:
        "results/multiqc_report.html",
        "results/de_results.csv"

rule fastp_trim:
    input:
        r1 = "data/{sample}_R1.fq.gz",
        r2 = "data/{sample}_R2.fq.gz"
    output:
        r1 = "trimmed/{sample}_R1.fq.gz",
        r2 = "trimmed/{sample}_R2.fq.gz",
        json = "qc/{sample}_fastp.json",
        html = "qc/{sample}_fastp.html"
    threads: 4
    resources:
        mem_mb = lambda wildcards, attempt: 4000 * attempt,   # escalate RAM on an OOM retry
        runtime = 60                                          # MINUTES, not seconds
    retries: 2
    container:
        "docker://quay.io/biocontainers/fastp:0.23.4--hadf994f_2"   # pinned; swap for @sha256: digest for full repro
    log:
        "logs/fastp/{sample}.log"
    shell:
        """
        fastp -i {input.r1} -I {input.r2} \
            -o {output.r1} -O {output.r2} \
            --json {output.json} --html {output.html} \
            --thread {threads} 2> {log}
        """

rule salmon_quant:
    input:
        r1 = "trimmed/{sample}_R1.fq.gz",
        r2 = "trimmed/{sample}_R2.fq.gz",
        index = SALMON_INDEX
    output:
        directory("salmon/{sample}")
    threads: 8
    resources:
        mem_mb = lambda wildcards, attempt: 8000 * attempt,
        runtime = 120
    retries: 2
    container:
        "docker://quay.io/biocontainers/salmon:1.10.2--h6dccd9a_2"
    log:
        "logs/salmon/{sample}.log"
    shell:
        """
        salmon quant -i {input.index} -l A \
            -1 {input.r1} -2 {input.r2} \
            -o {output} --threads {threads} 2> {log}
        """

rule multiqc:
    input:
        fastp = expand("qc/{sample}_fastp.json", sample=SAMPLES),
        salmon = expand("salmon/{sample}", sample=SAMPLES)
    output:
        "results/multiqc_report.html"
    container:
        "docker://quay.io/biocontainers/multiqc:1.21--pyhdfd78af_0"
    log:
        "logs/multiqc.log"
    shell:
        """
        multiqc qc/ salmon/ -o results/ -n multiqc_report 2> {log}
        """

rule deseq2_analysis:
    input:
        quants = expand("salmon/{sample}", sample=SAMPLES),
        metadata = config["metadata"]
    output:
        results = "results/de_results.csv",
        normalized = "results/normalized_counts.csv"
    log:
        "logs/deseq2.log"
    script:
        "scripts/run_deseq2.R"