End-to-end genome assembly workflow from reads to polished assembly with QC. Supports short reads (SPAdes), long reads (Flye), and hybrid approaches. Use when assembling genomes from raw reads.
Complete workflow from sequencing reads to polished, quality-assessed genome assembly.
Reads (short and/or long)
|
v
[1. QC & Filtering] -----> fastp, NanoPlot
|
v
[2. Assembly] -----------> SPAdes (short) or Flye (long)
|
v
[3. Polishing] ----------> Pilon (short) or medaka (long)
|
v
[4. QC Assessment] ------> QUAST, BUSCO
|
v
Final polished assembly
fastp -i reads_R1.fastq.gz -I reads_R2.fastq.gz \
-o trimmed_R1.fq.gz -O trimmed_R2.fq.gz \
--detect_adapter_for_pe \
--qualified_quality_phred 20 \
--length_required 50 \
--html qc_report.html
# Standard bacterial assembly
spades.py \
-1 trimmed_R1.fq.gz \
-2 trimmed_R2.fq.gz \
-o spades_output \
--careful \
-t 16 \
-m 64
# For isolate genomes
spades.py --isolate \
-1 trimmed_R1.fq.gz \
-2 trimmed_R2.fq.gz \
-o spades_output \
-t 16
# Align reads to assembly
bwa index spades_output/scaffolds.fasta
bwa mem -t 16 spades_output/scaffolds.fasta \
trimmed_R1.fq.gz trimmed_R2.fq.gz | \
samtools sort -@ 4 -o aligned.bam
samtools index aligned.bam
# Polish
pilon --genome spades_output/scaffolds.fasta \
--frags aligned.bam \
--output polished \
--threads 16
# NanoPlot for long-read QC
NanoPlot --fastq reads.fastq.gz \
--outdir nanoplot_output \
--threads 8
# ONT raw reads
flye --nano-raw reads.fastq.gz \
--out-dir flye_output \
--threads 16 \
--genome-size 5m
# ONT HQ reads (sup/dna_r10)
flye --nano-hq reads.fastq.gz \
--out-dir flye_output \
--threads 16 \
--genome-size 5m
# PacBio HiFi
flye --pacbio-hifi reads.fastq.gz \
--out-dir flye_output \
--threads 16 \
--genome-size 5m
# Polish with medaka (for ONT)
medaka_consensus \
-i reads.fastq.gz \
-d flye_output/assembly.fasta \
-o medaka_output \
-t 16 \
-m r1041_e82_400bps_sup_v4.3.0 # Match your basecalling model
# Flye with long reads, then polish with short reads
flye --nano-hq long_reads.fastq.gz \
--out-dir flye_output \
--threads 16 \
--genome-size 5m
# Polish with short reads using Pilon
bwa index flye_output/assembly.fasta
bwa mem -t 16 flye_output/assembly.fasta \
short_R1.fq.gz short_R2.fq.gz | \
samtools sort -@ 4 -o aligned.bam
samtools index aligned.bam
pilon --genome flye_output/assembly.fasta \
--frags aligned.bam \
--output hybrid_polished \
--threads 16
quast.py polished.fasta \
-r reference.fasta \
-g genes.gff \
-o quast_output \
-t 8
# Without reference
quast.py polished.fasta \
-o quast_output \
-t 8
# Download lineage database
busco --download bacteria_odb10
# Run BUSCO
busco -i polished.fasta \
-l bacteria_odb10 \
-o busco_output \
-m genome \
-c 8
| Tool | Parameter | Bacteria | Eukaryote |
|---|---|---|---|
| SPAdes | --careful | Yes | Optional |
| SPAdes | -m | 64GB | 256GB+ |
| Flye | --genome-size | 5m | Species-specific |
| Flye | --meta | If metagenome | No |
| BUSCO | -l | bacteria_odb10 | eukaryota_odb10 |
| Issue | Likely Cause | Solution |
|---|---|---|
| Fragmented assembly | Low coverage, repetitive genome | Increase coverage, use long reads |
| Low N50 | Short reads only | Add long reads for scaffolding |
| Low BUSCO | Incomplete assembly, wrong lineage | Check coverage, try different lineage |
| Assembly too large | Contamination, heterozygosity | Filter reads, check for contamination |
#!/bin/bash
set -e
THREADS=16
GENOME_SIZE="5m"
LONG_READS="long_reads.fastq.gz"
SHORT_R1="short_R1.fastq.gz"
SHORT_R2="short_R2.fastq.gz"
BUSCO_LINEAGE="bacteria_odb10"
OUTDIR="assembly_results"
mkdir -p ${OUTDIR}/{qc,assembly,polished,quast,busco}
# Step 1: QC
echo "=== QC ==="
NanoPlot --fastq ${LONG_READS} --outdir ${OUTDIR}/qc/nanoplot -t ${THREADS}
fastp -i ${SHORT_R1} -I ${SHORT_R2} \
-o ${OUTDIR}/qc/short_R1.fq.gz -O ${OUTDIR}/qc/short_R2.fq.gz \
--html ${OUTDIR}/qc/fastp.html
# Step 2: Assembly with Flye
echo "=== Assembly ==="
flye --nano-hq ${LONG_READS} \
--out-dir ${OUTDIR}/assembly \
--threads ${THREADS} \
--genome-size ${GENOME_SIZE}
# Step 3: Polish with short reads
echo "=== Polishing ==="
bwa index ${OUTDIR}/assembly/assembly.fasta
bwa mem -t ${THREADS} ${OUTDIR}/assembly/assembly.fasta \
${OUTDIR}/qc/short_R1.fq.gz ${OUTDIR}/qc/short_R2.fq.gz | \
samtools sort -@ 4 -o ${OUTDIR}/polished/aligned.bam
samtools index ${OUTDIR}/polished/aligned.bam
pilon --genome ${OUTDIR}/assembly/assembly.fasta \
--frags ${OUTDIR}/polished/aligned.bam \
--output ${OUTDIR}/polished/final \
--threads ${THREADS}
# Step 4: QC
echo "=== Quality Assessment ==="
quast.py ${OUTDIR}/polished/final.fasta -o ${OUTDIR}/quast -t ${THREADS}
busco -i ${OUTDIR}/polished/final.fasta -l ${BUSCO_LINEAGE} \
-o busco -m genome -c ${THREADS} --out_path ${OUTDIR}
echo "=== Assembly Complete ==="
echo "Final assembly: ${OUTDIR}/polished/final.fasta"
cat ${OUTDIR}/quast/report.txt