File size: 4,424 Bytes
5e7f7bf | 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 | #!/bin/bash
set -e
# =============================================================================
# Task 24: Multi-sample Variant Calling and Comparison
#
# DAG (depth 6, per-sample parallel then merge):
#
# L0: reads_A + reads_B + reference
# L1: fastp A fastp B
# L2: bwa A bwa B
# L3: freebayes A freebayes B
# L4: bcftools filter A bcftools filter B
# └──────────┬───────────┘
# L5: bcftools isec (shared vs unique variants)
# └── variant annotation + summary
# 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"
SAMPLES=("sampleA" "sampleB")
log_step() {
echo "=================================================================="
echo "STEP: $1"
echo "$(date)"
echo "=================================================================="
}
mkdir -p "${OUT}"/{trimmed,aligned,variants,filtered,comparison} "${RES}"
# Per-sample processing (L1-L4)
for SAMPLE in "${SAMPLES[@]}"; do
# L1: Trim
log_step "L1: fastp ${SAMPLE}"
if [ ! -f "${OUT}/trimmed/${SAMPLE}_R1.fastq.gz" ]; then
fastp --in1 "${DATA}/${SAMPLE}_R1.fastq.gz" --in2 "${DATA}/${SAMPLE}_R2.fastq.gz" \
--out1 "${OUT}/trimmed/${SAMPLE}_R1.fastq.gz" --out2 "${OUT}/trimmed/${SAMPLE}_R2.fastq.gz" \
--detect_adapter_for_pe --thread ${THREADS} \
--json "${OUT}/trimmed/${SAMPLE}_fastp.json"
else echo "Skipping (exists)"; fi
# L2: Align
log_step "L2: bwa ${SAMPLE}"
if [ ! -f "${OUT}/aligned/${SAMPLE}.bam" ]; then
[ ! -f "${REFERENCE}.bwt" ] && bwa index "${REFERENCE}" 2>/dev/null
bwa mem -t ${THREADS} -R "@RG\tID:${SAMPLE}\tSM:${SAMPLE}\tPL:ILLUMINA" \
"${REFERENCE}" "${OUT}/trimmed/${SAMPLE}_R1.fastq.gz" "${OUT}/trimmed/${SAMPLE}_R2.fastq.gz" \
| samtools sort -@ ${THREADS} -o "${OUT}/aligned/${SAMPLE}.bam"
samtools index "${OUT}/aligned/${SAMPLE}.bam"
else echo "Skipping (exists)"; fi
# L3: Call variants
log_step "L3: freebayes ${SAMPLE}"
if [ ! -f "${OUT}/variants/${SAMPLE}.vcf" ]; then
freebayes -f "${REFERENCE}" "${OUT}/aligned/${SAMPLE}.bam" \
--min-mapping-quality 20 --min-base-quality 20 \
> "${OUT}/variants/${SAMPLE}.vcf"
else echo "Skipping (exists)"; fi
# L4: Filter
log_step "L4: bcftools filter ${SAMPLE}"
if [ ! -f "${OUT}/filtered/${SAMPLE}.vcf.gz" ]; then
bcftools filter -i 'QUAL>20 && INFO/DP>5' "${OUT}/variants/${SAMPLE}.vcf" \
| bcftools view -Oz -o "${OUT}/filtered/${SAMPLE}.vcf.gz"
bcftools index "${OUT}/filtered/${SAMPLE}.vcf.gz"
else echo "Skipping (exists)"; fi
done
# L5: Compare variants between samples
log_step "L5: bcftools isec (shared vs unique)"
if [ ! -d "${OUT}/comparison/isec" ]; then
bcftools isec -p "${OUT}/comparison/isec" \
"${OUT}/filtered/sampleA.vcf.gz" \
"${OUT}/filtered/sampleB.vcf.gz"
else echo "Skipping (exists)"; fi
# Parse comparison results
UNIQUE_A=$(grep -cv "^#" "${OUT}/comparison/isec/0000.vcf" 2>/dev/null || echo "0")
UNIQUE_B=$(grep -cv "^#" "${OUT}/comparison/isec/0001.vcf" 2>/dev/null || echo "0")
SHARED=$(grep -cv "^#" "${OUT}/comparison/isec/0002.vcf" 2>/dev/null || echo "0")
# Per-sample stats
VARS_A=$(bcftools stats "${OUT}/filtered/sampleA.vcf.gz" | grep "^SN" | grep "number of records" | cut -f4)
VARS_B=$(bcftools stats "${OUT}/filtered/sampleB.vcf.gz" | grep "^SN" | grep "number of records" | cut -f4)
SNPS_A=$(bcftools stats "${OUT}/filtered/sampleA.vcf.gz" | grep "^SN" | grep "number of SNPs" | cut -f4)
SNPS_B=$(bcftools stats "${OUT}/filtered/sampleB.vcf.gz" | grep "^SN" | grep "number of SNPs" | cut -f4)
# L6: MERGE
log_step "L6-MERGE"
cat > "${RES}/variant_comparison.csv" << CSVEOF
metric,value
total_variants_sampleA,${VARS_A}
total_variants_sampleB,${VARS_B}
snps_sampleA,${SNPS_A}
snps_sampleB,${SNPS_B}
unique_to_sampleA,${UNIQUE_A}
unique_to_sampleB,${UNIQUE_B}
shared_variants,${SHARED}
CSVEOF
echo ""
echo "=== Pipeline complete ==="
cat "${RES}/variant_comparison.csv"
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