File size: 4,699 Bytes
ff6001d | 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 | #!/bin/bash
set -uo pipefail
# =============================================================================
# Task: DDA Label-Free Quantitative Proteomics
#
# DAG structure (depth 10, 3 convergence points):
#
# L0: mzML mass spec file + protein FASTA database
# L1: DecoyDatabase (add reverse decoys)
# L2: PeakPickerHiRes (centroid if profile mode)
# L3: CometAdapter (database search — peptide-spectrum matching)
# L4: PeptideIndexer (map peptides to protein sequences)
# L5: FalseDiscoveryRate (target-decoy FDR estimation)
# ├──────────────────────────────────────────────────┐
# L6: IDFilter (1% PSM FDR) FDR statistics
# │ │
# L7: TextExporter (idXML → TSV) QC metrics
# │ │
# L8: Parse protein/peptide/PSM counts ◄────────────────┘ [CONVERGENCE 1+2]
# L9: MERGE [CONVERGENCE 3]
# =============================================================================
THREADS=$(( $(nproc) > 8 ? 8 : $(nproc) ))
SCRIPT_DIR="$(cd "$(dirname "${BASH_SOURCE[0]}")" && pwd)"
DATA="${SCRIPT_DIR}/data"
REF="${SCRIPT_DIR}/reference"
OUT="${SCRIPT_DIR}/outputs"
RES="${SCRIPT_DIR}/results"
MZML="${DATA}/sample.mzML"
DATABASE="${REF}/database.fasta"
log_step() { echo "== STEP: $1 == $(date)"; }
mkdir -p "${OUT}"/{decoy,picked,search_comet,indexed,fdr,filtered,quant} "${RES}"
# L1: Add decoy sequences
log_step "L1: DecoyDatabase"
if [ ! -f "${OUT}/decoy/target_decoy.fasta" ]; then
DecoyDatabase -in "${DATABASE}" -out "${OUT}/decoy/target_decoy.fasta" \
-decoy_string "DECOY_" -decoy_string_position prefix
fi
# L2: Peak picking
log_step "L2: PeakPickerHiRes"
if [ ! -f "${OUT}/picked/picked.mzML" ]; then
PeakPickerHiRes -in "${MZML}" -out "${OUT}/picked/picked.mzML" \
-algorithm:ms_levels 1 2 2>/dev/null || {
echo "Data already centroided, using as-is"
cp "${MZML}" "${OUT}/picked/picked.mzML"
}
fi
# L3: Comet database search
log_step "L3: CometAdapter"
if [ ! -f "${OUT}/search_comet/comet.idXML" ]; then
CometAdapter -in "${OUT}/picked/picked.mzML" \
-database "${OUT}/decoy/target_decoy.fasta" \
-out "${OUT}/search_comet/comet.idXML" \
-precursor_mass_tolerance 10 \
-precursor_error_units ppm \
-fragment_mass_tolerance 0.02 \
-threads ${THREADS} 2>&1 || true
fi
# L4: PeptideIndexer
log_step "L4: PeptideIndexer"
if [ ! -f "${OUT}/indexed/indexed.idXML" ]; then
PeptideIndexer -in "${OUT}/search_comet/comet.idXML" \
-fasta "${OUT}/decoy/target_decoy.fasta" \
-out "${OUT}/indexed/indexed.idXML" \
-decoy_string "DECOY_" -decoy_string_position prefix \
-enzyme:name Trypsin
fi
# L5: FDR
log_step "L5: FalseDiscoveryRate"
if [ ! -f "${OUT}/fdr/fdr.idXML" ]; then
FalseDiscoveryRate -in "${OUT}/indexed/indexed.idXML" \
-out "${OUT}/fdr/fdr.idXML" -force
fi
# L6: Filter at 1% FDR
log_step "L6: IDFilter"
if [ ! -f "${OUT}/filtered/filtered.idXML" ]; then
IDFilter -in "${OUT}/fdr/fdr.idXML" \
-out "${OUT}/filtered/filtered.idXML" \
-score:psm 0.01
fi
# L7: Export
log_step "L7: TextExporter"
if [ ! -f "${OUT}/quant/results.tsv" ]; then
TextExporter -in "${OUT}/filtered/filtered.idXML" \
-out "${OUT}/quant/results.tsv"
fi
# MERGE
log_step "MERGE"
SPECTRA=$(grep -c "<spectrum " "${OUT}/picked/picked.mzML" 2>/dev/null || true)
SPECTRA=${SPECTRA:-0}
DB_SIZE=$(grep -c "^>" "${OUT}/decoy/target_decoy.fasta" 2>/dev/null || true)
DB_SIZE=${DB_SIZE:-0}
SEARCH_HITS=$(grep -c "PeptideHit" "${OUT}/search_comet/comet.idXML" 2>/dev/null || true)
SEARCH_HITS=${SEARCH_HITS:-0}
PSMS=$(grep -c "^PEPTIDE" "${OUT}/quant/results.tsv" 2>/dev/null || true)
PSMS=${PSMS:-0}
PEPTIDES=$(grep "^PEPTIDE" "${OUT}/quant/results.tsv" 2>/dev/null | awk '{print $2}' | sort -u | wc -l | tr -d ' ')
PROTEINS=$(grep -c "^PROTEIN" "${OUT}/quant/results.tsv" 2>/dev/null || true)
PROTEINS=${PROTEINS:-0}
cat > "${RES}/proteomics_report.csv" << CSVEOF
metric,value
total_spectra,${SPECTRA}
database_size_with_decoys,${DB_SIZE}
search_engine_hits,${SEARCH_HITS}
psms_after_fdr,${PSMS}
unique_peptides,${PEPTIDES}
proteins_identified,${PROTEINS}
CSVEOF
echo ""
echo "=== Pipeline complete ==="
cat "${RES}/proteomics_report.csv"
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