File size: 7,929 Bytes
0562e15 | 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 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 | #!/bin/bash
set -e
# =============================================================================
# Task 16: Long-read de novo Assembly and Polishing
#
# DAG (depth 7, linear chain with late fan-out):
#
# L0: raw nanopore reads
# L1: NanoPlot (read QC)
# L2: Filtlong (quality/length filter)
# L3: Flye (de novo assembly)
# L4: minimap2+samtools (map reads → sorted BAM for polishing)
# L5: Medaka (consensus polishing, round 1)
# ├──────────────────────────────────────┐
# L6: QUAST (assembly QC vs ref) Prokka (annotation)
# │ │
# ├── BUSCO (completeness) abricate (AMR)
# │ │
# L7: 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"
READS="${DATA}/barcode10.fastq.gz"
REFERENCE="${REF}/salmonella_ref.fna"
log_step() {
echo "=================================================================="
echo "STEP: $1"
echo "$(date)"
echo "=================================================================="
}
mkdir -p "${OUT}"/{nanoplot,filtered,assembly,mapping,polished,qc,prokka,amr} "${RES}"
# ===========================================================================
# L1: Read QC with NanoPlot
# ===========================================================================
log_step "L1: NanoPlot read QC"
if [ ! -f "${OUT}/nanoplot/NanoStats.txt" ]; then
NanoPlot --fastq "${READS}" -o "${OUT}/nanoplot" -t ${THREADS} --plots dot
else echo "Skipping (exists)"; fi
# ===========================================================================
# L2: Quality filtering with Filtlong
# ===========================================================================
log_step "L2: Filtlong quality filtering"
if [ ! -f "${OUT}/filtered/filtered.fastq.gz" ]; then
filtlong --min_length 200 "${READS}" | gzip > "${OUT}/filtered/filtered.fastq.gz"
else echo "Skipping (exists)"; fi
# ===========================================================================
# L3: De novo assembly with Flye
# ===========================================================================
log_step "L3: Flye assembly"
if [ ! -f "${OUT}/assembly/assembly.fasta" ]; then
flye --nano-raw "${OUT}/filtered/filtered.fastq.gz" \
--out-dir "${OUT}/assembly" \
--threads ${THREADS} --genome-size 5m
else echo "Skipping (exists)"; fi
# ===========================================================================
# L4: Map reads back to assembly for polishing
# ===========================================================================
log_step "L4: minimap2 read mapping"
if [ ! -f "${OUT}/mapping/reads2assembly.bam" ]; then
minimap2 -ax map-ont -t ${THREADS} \
"${OUT}/assembly/assembly.fasta" "${OUT}/filtered/filtered.fastq.gz" \
| samtools sort -@ ${THREADS} -o "${OUT}/mapping/reads2assembly.bam"
samtools index "${OUT}/mapping/reads2assembly.bam"
else echo "Skipping (exists)"; fi
# ===========================================================================
# L5: Consensus polishing with Medaka
# ===========================================================================
log_step "L5: Medaka polishing"
if [ ! -f "${OUT}/polished/consensus.fasta" ]; then
medaka_consensus -i "${OUT}/filtered/filtered.fastq.gz" \
-d "${OUT}/assembly/assembly.fasta" \
-o "${OUT}/polished" \
-t ${THREADS} -m r1041_e82_400bps_sup_v5.0.0 2>&1 || {
echo "WARNING: Medaka failed with specified model, trying default"
medaka_consensus -i "${OUT}/filtered/filtered.fastq.gz" \
-d "${OUT}/assembly/assembly.fasta" \
-o "${OUT}/polished" \
-t ${THREADS} 2>&1 || {
echo "WARNING: Medaka failed, using unpolished assembly"
cp "${OUT}/assembly/assembly.fasta" "${OUT}/polished/consensus.fasta"
}
}
else echo "Skipping (exists)"; fi
FINAL="${OUT}/polished/consensus.fasta"
# ===========================================================================
# L6 LEFT: Assembly QC
# ===========================================================================
log_step "L6-LEFT: QUAST assembly QC"
if [ ! -f "${OUT}/qc/quast/report.tsv" ]; then
quast "${FINAL}" -r "${REFERENCE}" -o "${OUT}/qc/quast" -t ${THREADS}
else echo "Skipping (exists)"; fi
log_step "L6-LEFT: BUSCO completeness"
if [ ! -d "${OUT}/qc/busco" ]; then
busco -i "${FINAL}" -o busco --out_path "${OUT}/qc" \
-l bacteria_odb10 -m genome -c ${THREADS} --force
else echo "Skipping (exists)"; fi
# ===========================================================================
# L6 RIGHT: Annotation + AMR
# ===========================================================================
log_step "L6-RIGHT: Prokka annotation"
if [ ! -f "${OUT}/prokka/SALM.gff" ]; then
prokka "${FINAL}" --outdir "${OUT}/prokka" --prefix SALM \
--cpus ${THREADS} --kingdom Bacteria --genus Salmonella --force
else echo "Skipping (exists)"; fi
log_step "L6-RIGHT: abricate AMR detection"
if [ ! -f "${OUT}/amr/abricate_card.tsv" ]; then
abricate "${FINAL}" --db card --minid 80 --mincov 60 > "${OUT}/amr/abricate_card.tsv"
else echo "Skipping (exists)"; fi
# ===========================================================================
# L7: MERGE
# ===========================================================================
log_step "L7-MERGE: Building results"
# Read QC stats
MEAN_LEN=$(grep "Mean read length" "${OUT}/nanoplot/NanoStats.txt" | awk '{print $NF}' | tr -d ',')
MEAN_QUAL=$(grep "Mean read quality" "${OUT}/nanoplot/NanoStats.txt" | awk '{print $NF}')
TOTAL_BASES=$(grep "Total bases" "${OUT}/nanoplot/NanoStats.txt" | awk '{print $NF}' | tr -d ',')
NUM_READS=$(grep "Number of reads" "${OUT}/nanoplot/NanoStats.txt" | head -1 | awk '{print $NF}' | tr -d ',')
# Assembly stats from QUAST
TOTAL_LEN=$(grep "^Total length" "${OUT}/qc/quast/report.tsv" | head -1 | cut -f2)
NUM_CONTIGS=$(grep "^# contigs " "${OUT}/qc/quast/report.tsv" | head -1 | cut -f2)
N50=$(grep "^N50" "${OUT}/qc/quast/report.tsv" | cut -f2)
GC=$(grep "^GC" "${OUT}/qc/quast/report.tsv" | cut -f2)
LARGEST=$(grep "^Largest contig" "${OUT}/qc/quast/report.tsv" | cut -f2)
GENOME_FRAC=$(grep "^Genome fraction" "${OUT}/qc/quast/report.tsv" | cut -f2)
MISASSEMBLIES=$(grep "^# misassemblies" "${OUT}/qc/quast/report.tsv" | cut -f2)
# BUSCO
BUSCO_SUM=$(grep "C:" "${OUT}/qc/busco/short_summary.specific.bacteria_odb10.busco.txt" 2>/dev/null \
| head -1 | sed 's/^[[:space:]]*//;s/[[:space:]]*$//' || echo "N/A")
# Prokka
CDS=$(grep "^CDS" "${OUT}/prokka/SALM.txt" | awk '{print $2}')
TRNA=$(grep "^tRNA" "${OUT}/prokka/SALM.txt" | awk '{print $2}')
RRNA=$(grep "^rRNA" "${OUT}/prokka/SALM.txt" | awk '{print $2}')
# AMR
AMR_COUNT=$(tail -n +2 "${OUT}/amr/abricate_card.tsv" 2>/dev/null | wc -l | tr -d ' ')
cat > "${RES}/longread_assembly_report.csv" << CSVEOF
metric,value
num_reads,${NUM_READS}
mean_read_length,${MEAN_LEN}
mean_read_quality,${MEAN_QUAL}
total_bases,${TOTAL_BASES}
assembly_length,${TOTAL_LEN}
num_contigs,${NUM_CONTIGS}
n50,${N50}
gc_content,${GC}
largest_contig,${LARGEST}
genome_fraction,${GENOME_FRAC}
misassemblies,${MISASSEMBLIES}
completeness,${BUSCO_SUM}
cds_count,${CDS}
trna_count,${TRNA}
rrna_count,${RRNA}
amr_genes,${AMR_COUNT}
CSVEOF
echo ""
echo "=== Pipeline complete ==="
cat "${RES}/longread_assembly_report.csv"
echo ""
ls -lh "${RES}/"
|