#!/bin/bash set -e # ============================================================================= # Task 23: Genome Coverage and Mapping Quality Analysis # # DAG (depth 5): # # L0: PE reads + reference # L1: fastp (trim) # L2: bwa mem (align) # ├──────────────────────────────────────┐ # L3: samtools stats/flagstat mosdepth (coverage) # │ │ # L4: samtools idxstats ├── per-base coverage # (per-contig mapping) └── per-window coverage # │ │ # └── bedtools genomecov deeptools bamCoverage # (coverage histogram) (normalized signal) # L5: MERGE # ============================================================================= 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" REFERENCE="${REF}/reference.fna" log_step() { echo "==================================================================" echo "STEP: $1" echo "$(date)" echo "==================================================================" } mkdir -p "${OUT}"/{trimmed,aligned,stats,coverage} "${RES}" # =========================================================================== # L1: Trimming # =========================================================================== log_step "L1: fastp" if [ ! -f "${OUT}/trimmed/R1.fastq" ]; then fastp --in1 "${DATA}/reads_R1.fastq" --in2 "${DATA}/reads_R2.fastq" \ --out1 "${OUT}/trimmed/R1.fastq" --out2 "${OUT}/trimmed/R2.fastq" \ --detect_adapter_for_pe --thread ${THREADS} \ --json "${OUT}/trimmed/fastp.json" else echo "Skipping (exists)"; fi # =========================================================================== # L2: Alignment # =========================================================================== log_step "L2: bwa mem" if [ ! -f "${OUT}/aligned/reads.sorted.bam" ]; then bwa index "${REFERENCE}" 2>/dev/null bwa mem -t ${THREADS} -R "@RG\tID:sample\tSM:sample\tPL:ILLUMINA" \ "${REFERENCE}" "${OUT}/trimmed/R1.fastq" "${OUT}/trimmed/R2.fastq" \ | samtools sort -@ ${THREADS} -o "${OUT}/aligned/reads.sorted.bam" samtools index "${OUT}/aligned/reads.sorted.bam" else echo "Skipping (exists)"; fi BAM="${OUT}/aligned/reads.sorted.bam" # =========================================================================== # L3-A: samtools stats + flagstat # =========================================================================== log_step "L3-A: samtools stats" if [ ! -f "${OUT}/stats/samtools_stats.txt" ]; then samtools stats "${BAM}" > "${OUT}/stats/samtools_stats.txt" samtools flagstat "${BAM}" > "${OUT}/stats/flagstat.txt" samtools idxstats "${BAM}" > "${OUT}/stats/idxstats.txt" else echo "Skipping (exists)"; fi # =========================================================================== # L3-B: mosdepth coverage analysis # =========================================================================== log_step "L3-B: mosdepth" if [ ! -f "${OUT}/coverage/sample.mosdepth.summary.txt" ]; then mosdepth -t ${THREADS} --by 1000 "${OUT}/coverage/sample" "${BAM}" else echo "Skipping (exists)"; fi # =========================================================================== # L4-A: bedtools genomecov (coverage histogram) # =========================================================================== log_step "L4-A: bedtools genomecov" if [ ! -f "${OUT}/coverage/genomecov.txt" ]; then bedtools genomecov -ibam "${BAM}" > "${OUT}/coverage/genomecov.txt" else echo "Skipping (exists)"; fi # =========================================================================== # L4-B: deeptools bamCoverage (normalized signal) # =========================================================================== log_step "L4-B: deeptools bamCoverage" if [ ! -f "${OUT}/coverage/sample.bw" ]; then bamCoverage --bam "${BAM}" --outFileName "${OUT}/coverage/sample.bw" \ --binSize 100 --normalizeUsing RPGC \ --effectiveGenomeSize 2900000 \ --numberOfProcessors ${THREADS} 2>/dev/null || true else echo "Skipping (exists)"; fi # =========================================================================== # L5: MERGE # =========================================================================== log_step "L5-MERGE" # Parse samtools stats TOTAL_READS=$(grep "^SN" "${OUT}/stats/samtools_stats.txt" | grep "raw total sequences" | cut -f3) MAPPED_READS=$(grep "^SN" "${OUT}/stats/samtools_stats.txt" | grep "reads mapped:" | cut -f3) MAPPED_PCT=$(grep "mapped (" "${OUT}/stats/flagstat.txt" | head -1 | grep -oP '[\d.]+%' | tr -d '%') PROPERLY_PAIRED=$(grep "properly paired" "${OUT}/stats/flagstat.txt" | grep -oP '[\d.]+%' | tr -d '%') AVG_QUALITY=$(grep "^SN" "${OUT}/stats/samtools_stats.txt" | grep "average quality" | cut -f3) AVG_INSERT=$(grep "^SN" "${OUT}/stats/samtools_stats.txt" | grep "insert size average" | cut -f3) ERROR_RATE=$(grep "^SN" "${OUT}/stats/samtools_stats.txt" | grep "error rate" | cut -f3) # Parse mosdepth MEAN_COV=$(awk '$1=="total" {print $4}' "${OUT}/coverage/sample.mosdepth.summary.txt") MIN_COV=$(awk '$1=="total" {print $5}' "${OUT}/coverage/sample.mosdepth.summary.txt") MAX_COV=$(awk '$1=="total" {print $6}' "${OUT}/coverage/sample.mosdepth.summary.txt") # Coverage breadth from genomecov (% bases with >=1x coverage) BREADTH=$(awk '$1=="genome" && $2==0 {print 100-$5*100}' "${OUT}/coverage/genomecov.txt" | head -1) cat > "${RES}/mapping_qc_report.csv" << CSVEOF metric,value total_reads,${TOTAL_READS} mapped_reads,${MAPPED_READS} mapping_rate,${MAPPED_PCT} properly_paired_rate,${PROPERLY_PAIRED} average_base_quality,${AVG_QUALITY} average_insert_size,${AVG_INSERT} error_rate,${ERROR_RATE} mean_coverage,${MEAN_COV} min_coverage,${MIN_COV} max_coverage,${MAX_COV} coverage_breadth_pct,${BREADTH} CSVEOF echo "" echo "=== Pipeline complete ===" cat "${RES}/mapping_qc_report.csv" echo "" ls -lh "${RES}/"