#!/bin/bash set -e # ============================================================================= # Task 25: Consensus Genome Generation # # DAG (depth 6): # # L0: reads + reference # L1: fastp (trim) # L2: bwa mem (align) # L3: ├── samtools mpileup → bcftools call (variant calling) # └── mosdepth (coverage for masking) # L4: bcftools consensus (apply variants to reference) # + bedtools (mask low-coverage regions) # L5: ├── seqkit stats (consensus QC) # └── nucmer (compare consensus vs reference) # L6: 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 "${OUT}"/{trimmed,aligned,variants,coverage,consensus,comparison} "${RES}" # L1 log_step "L1: fastp" if [ ! -f "${OUT}/trimmed/R1.fastq.gz" ]; then fastp --in1 "${DATA}/reads_R1.fastq.gz" --in2 "${DATA}/reads_R2.fastq.gz" \ --out1 "${OUT}/trimmed/R1.fastq.gz" --out2 "${OUT}/trimmed/R2.fastq.gz" \ --detect_adapter_for_pe --thread ${THREADS} --json "${OUT}/trimmed/fastp.json" else echo "Skipping (exists)"; fi # L2 log_step "L2: bwa" if [ ! -f "${OUT}/aligned/reads.bam" ]; then [ ! -f "${REFERENCE}.bwt" ] && bwa index "${REFERENCE}" 2>/dev/null bwa mem -t ${THREADS} -R "@RG\tID:sample\tSM:sample\tPL:ILLUMINA" \ "${REFERENCE}" "${OUT}/trimmed/R1.fastq.gz" "${OUT}/trimmed/R2.fastq.gz" \ | samtools sort -@ ${THREADS} -o "${OUT}/aligned/reads.bam" samtools index "${OUT}/aligned/reads.bam" else echo "Skipping (exists)"; fi # L3-A: Variant calling log_step "L3-A: bcftools mpileup + call" if [ ! -f "${OUT}/variants/calls.vcf.gz" ]; then samtools faidx "${REFERENCE}" 2>/dev/null || true bcftools mpileup -f "${REFERENCE}" "${OUT}/aligned/reads.bam" --max-depth 500 -Q 20 \ | bcftools call -c --ploidy 1 -Oz -o "${OUT}/variants/calls.vcf.gz" bcftools index "${OUT}/variants/calls.vcf.gz" else echo "Skipping (exists)"; fi # L3-B: Coverage for masking log_step "L3-B: mosdepth coverage" if [ ! -f "${OUT}/coverage/sample.per-base.bed.gz" ]; then mosdepth -t ${THREADS} "${OUT}/coverage/sample" "${OUT}/aligned/reads.bam" else echo "Skipping (exists)"; fi # L4: Generate consensus with low-coverage masking log_step "L4: consensus generation" if [ ! -f "${OUT}/consensus/consensus.fasta" ]; then # Create mask for regions with <5x coverage zcat "${OUT}/coverage/sample.per-base.bed.gz" | awk '$4 < 5' > "${OUT}/coverage/low_cov.bed" # Apply variants and mask bcftools consensus -f "${REFERENCE}" "${OUT}/variants/calls.vcf.gz" \ -m "${OUT}/coverage/low_cov.bed" \ > "${OUT}/consensus/consensus.fasta" else echo "Skipping (exists)"; fi # L5-A: Consensus QC log_step "L5-A: seqkit stats" seqkit stats "${OUT}/consensus/consensus.fasta" -T > "${OUT}/consensus/stats.tsv" # L5-B: Compare consensus vs reference log_step "L5-B: nucmer comparison" if [ ! -f "${OUT}/comparison/dnadiff.report" ]; then nucmer --prefix="${OUT}/comparison/alignment" "${REFERENCE}" "${OUT}/consensus/consensus.fasta" dnadiff -d "${OUT}/comparison/alignment.delta" -p "${OUT}/comparison/dnadiff" else echo "Skipping (exists)"; fi # L6: MERGE log_step "L6-MERGE" CONS_LEN=$(awk '/^>/{if(l)s+=l;l=0;next}{l+=length}END{s+=l;print s}' "${OUT}/consensus/consensus.fasta") REF_LEN=$(awk '/^>/{if(l)s+=l;l=0;next}{l+=length}END{s+=l;print s}' "${REFERENCE}") N_COUNT=$(grep -o "N" "${OUT}/consensus/consensus.fasta" | wc -l | tr -d ' ') N_PCT=$(python3 -c "print(f'{${N_COUNT}/${CONS_LEN}*100:.2f}')") IDENTITY=$(grep "AvgIdentity" "${OUT}/comparison/dnadiff.report" | head -1 | awk '{print $2}') ALIGNED=$(grep "AlignedBases" "${OUT}/comparison/dnadiff.report" | head -1 | awk '{print $2}' | sed 's/(.*//') SNPS_VS_REF=$(grep "TotalSNPs" "${OUT}/comparison/dnadiff.report" | head -1 | awk '{print $2}') MEAN_COV=$(awk '$1=="total" {print $4}' "${OUT}/coverage/sample.mosdepth.summary.txt") LOW_COV_BASES=$(wc -l < "${OUT}/coverage/low_cov.bed" | tr -d ' ') cat > "${RES}/consensus_report.csv" << CSVEOF metric,value reference_length,${REF_LEN} consensus_length,${CONS_LEN} masked_bases_n,${N_COUNT} masked_pct,${N_PCT} mean_coverage,${MEAN_COV} low_coverage_regions,${LOW_COV_BASES} identity_to_reference,${IDENTITY} aligned_bases,${ALIGNED} snps_vs_reference,${SNPS_VS_REF} CSVEOF cp "${OUT}/consensus/consensus.fasta" "${RES}/" echo "" echo "=== Pipeline complete ===" cat "${RES}/consensus_report.csv"