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tasks/longread-assembly/run_script.sh

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+#!/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}/"