File size: 5,956 Bytes
8733f82 | 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 | #!/bin/bash
set -e
# =============================================================================
# Task 19: Bacterial Pan-genome and Evolutionary Analysis
#
# DAG (depth 6, fan-out from annotation):
#
# L0: 5 E. coli genome assemblies
# L1: Prokka annotation (×5 parallel)
# L2: ├── Panaroo (pan-genome from 5 GFFs)
# │ ├── L3: core gene alignment
# │ │ └── L4: snp-sites → iqtree (core phylogeny)
# │ └── L3: gene presence/absence matrix
# │ └── L4: accessory genome stats
# └── mlst (×5)
# └── abricate (×5)
# L5: MERGE
# =============================================================================
THREADS=$(( $(nproc) > 8 ? 8 : $(nproc) ))
SCRIPT_DIR="$(cd "$(dirname "${BASH_SOURCE[0]}")" && pwd)"
DATA="${SCRIPT_DIR}/data"
OUT="${SCRIPT_DIR}/outputs"
RES="${SCRIPT_DIR}/results"
SAMPLES=("ecoli_K12" "ecoli_O157H7" "ecoli_CFT073" "ecoli_UTI89" "ecoli_APEC")
log_step() {
echo "=================================================================="
echo "STEP: $1"
echo "$(date)"
echo "=================================================================="
}
mkdir -p "${OUT}"/{prokka,mlst_out,amr,panaroo,phylogeny} "${RES}"
# ===========================================================================
# L0: Decompress genomes if needed
# ===========================================================================
log_step "L0: Prepare genomes"
for SAMPLE in "${SAMPLES[@]}"; do
if [ ! -f "${DATA}/${SAMPLE}.fna" ]; then
gunzip -k "${DATA}/${SAMPLE}.fna.gz" 2>/dev/null || true
fi
done
# ===========================================================================
# L1: Prokka annotation (×5)
# ===========================================================================
for SAMPLE in "${SAMPLES[@]}"; do
log_step "L1: Prokka ${SAMPLE}"
if [ ! -f "${OUT}/prokka/${SAMPLE}/${SAMPLE}.gff" ]; then
prokka "${DATA}/${SAMPLE}.fna" --outdir "${OUT}/prokka/${SAMPLE}" --prefix "${SAMPLE}" \
--cpus ${THREADS} --kingdom Bacteria --genus Escherichia --species coli --force
else echo "Skipping (exists)"; fi
done
# ===========================================================================
# L1 parallel: MLST + AMR (×5)
# ===========================================================================
echo "sample,scheme,sequence_type" > "${OUT}/mlst_out/mlst_all.csv"
for SAMPLE in "${SAMPLES[@]}"; do
log_step "L1: mlst + abricate ${SAMPLE}"
mlst "${DATA}/${SAMPLE}.fna" > "${OUT}/mlst_out/${SAMPLE}.tsv" 2>/dev/null || true
SCHEME=$(cut -f2 "${OUT}/mlst_out/${SAMPLE}.tsv" 2>/dev/null)
ST=$(cut -f3 "${OUT}/mlst_out/${SAMPLE}.tsv" 2>/dev/null)
echo "${SAMPLE},${SCHEME},${ST}" >> "${OUT}/mlst_out/mlst_all.csv"
if [ ! -f "${OUT}/amr/${SAMPLE}.tsv" ]; then
abricate "${DATA}/${SAMPLE}.fna" --db card --minid 80 --mincov 60 > "${OUT}/amr/${SAMPLE}.tsv" 2>/dev/null || true
fi
done
# ===========================================================================
# L2: Panaroo pan-genome
# ===========================================================================
log_step "L2: Panaroo"
if [ ! -f "${OUT}/panaroo/summary_statistics.txt" ]; then
GFF_FILES=""
for SAMPLE in "${SAMPLES[@]}"; do
GFF_FILES="${GFF_FILES} ${OUT}/prokka/${SAMPLE}/${SAMPLE}.gff"
done
panaroo -i ${GFF_FILES} -o "${OUT}/panaroo" --clean-mode strict \
-a core -c 0.98 --threads ${THREADS} 2>&1 || true
else echo "Skipping (exists)"; fi
# ===========================================================================
# L3-L4: Core gene phylogeny
# ===========================================================================
log_step "L3: snp-sites on core alignment"
if [ -f "${OUT}/panaroo/core_gene_alignment.aln" ] && [ ! -f "${OUT}/phylogeny/core_snps.fasta" ]; then
snp-sites -o "${OUT}/phylogeny/core_snps.fasta" "${OUT}/panaroo/core_gene_alignment.aln" 2>&1 || \
cp "${OUT}/panaroo/core_gene_alignment.aln" "${OUT}/phylogeny/core_snps.fasta"
fi
log_step "L4: iqtree phylogeny"
if [ -f "${OUT}/phylogeny/core_snps.fasta" ] && [ ! -f "${OUT}/phylogeny/core_tree.treefile" ]; then
iqtree -s "${OUT}/phylogeny/core_snps.fasta" -m GTR+G -bb 1000 \
-nt ${THREADS} --prefix "${OUT}/phylogeny/core_tree" 2>&1 || true
fi
# ===========================================================================
# L5: MERGE
# ===========================================================================
log_step "L5-MERGE"
CORE_GENES=$(grep "Core genes" "${OUT}/panaroo/summary_statistics.txt" 2>/dev/null | awk '{print $NF}' || echo "N/A")
TOTAL_GENES=$(grep "Total genes" "${OUT}/panaroo/summary_statistics.txt" 2>/dev/null | awk '{print $NF}' || echo "N/A")
SHELL_GENES=$(grep "Shell genes" "${OUT}/panaroo/summary_statistics.txt" 2>/dev/null | awk '{print $NF}' || echo "N/A")
CLOUD_GENES=$(grep "Cloud genes" "${OUT}/panaroo/summary_statistics.txt" 2>/dev/null | awk '{print $NF}' || echo "N/A")
cat > "${RES}/pangenome_report.csv" << CSVEOF
metric,value
num_genomes,${#SAMPLES[@]}
core_genes,${CORE_GENES}
shell_genes,${SHELL_GENES}
cloud_genes,${CLOUD_GENES}
total_genes,${TOTAL_GENES}
tree_available,$([ -f "${OUT}/phylogeny/core_tree.treefile" ] && echo "yes" || echo "no")
CSVEOF
cp "${OUT}/mlst_out/mlst_all.csv" "${RES}/" 2>/dev/null || true
cp "${OUT}/phylogeny/core_tree.treefile" "${RES}/core_phylogeny.nwk" 2>/dev/null || true
cp "${OUT}/panaroo/gene_presence_absence.csv" "${RES}/" 2>/dev/null || true
# Per-genome AMR summary
echo "sample,amr_genes" > "${RES}/amr_summary.csv"
for SAMPLE in "${SAMPLES[@]}"; do
COUNT=$(tail -n +2 "${OUT}/amr/${SAMPLE}.tsv" 2>/dev/null | wc -l | tr -d ' ')
echo "${SAMPLE},${COUNT}" >> "${RES}/amr_summary.csv"
done
echo ""
echo "=== Pipeline complete ==="
cat "${RES}/pangenome_report.csv"
echo ""
cat "${RES}/mlst_all.csv"
echo ""
ls -lh "${RES}/"
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