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Assembly QC

Evaluate genome assembly quality with contiguity metrics (QUAST) and gene completeness (BUSCO).

Key Metrics

| Metric | Good Assembly | |--------|---------------| | N50 | High (relative to genome) | | L50 | Low | | Contigs | Few | | Misassemblies | 0 (with reference) | | BUSCO Complete | >95% | | BUSCO Duplicated | <5% (unless polyploid) |

QUAST

Installation

bash
conda install -c bioconda quast

Basic Usage

bash
quast.py assembly.fasta -o quast_output

With Reference Genome

bash
quast.py assembly.fasta -r reference.fasta -o quast_output

Compare Multiple Assemblies

bash
quast.py assembly1.fa assembly2.fa assembly3.fa -o comparison

Key Options

| Option | Description | |--------|-------------| | -o | Output directory | | -r | Reference genome | | -g | Gene annotations (GFF) | | -t | Threads | | -m | Min contig length (default: 500) | | --large | For large genomes (>100Mb) | | --fragmented | For highly fragmented assemblies | | --scaffolds | Input is scaffolds (includes N-gaps) |

With Gene Annotations

bash
quast.py assembly.fasta -r reference.fasta -g genes.gff -o quast_output

For Large Genomes

bash
quast.py --large assembly.fasta -o quast_output -t 16

Output Files

quast_output/
├── report.txt        # Summary statistics
├── report.html       # Interactive report
├── report.tsv        # Tab-separated stats
├── icarus.html       # Contig viewer
└── aligned_stats/    # If reference provided

Key Output Metrics

| Metric | Description | |--------|-------------| | Total length | Sum of contig lengths | | # contigs | Number of contigs (>= min length) | | Largest contig | Length of largest contig | | N50 | 50% of assembly in contigs >= this length | | N90 | 90% of assembly in contigs >= this length | | L50 | Number of contigs comprising N50 | | GC % | GC content | | # misassemblies | With reference: structural errors | | Genome fraction | With reference: % of reference covered |

BUSCO

Installation

bash
conda install -c bioconda busco

Basic Usage

bash
busco -i assembly.fasta -m genome -l bacteria_odb10 -o busco_output

Key Options

| Option | Description | |--------|-------------| | -i | Input assembly | | -m | Mode: genome, proteins, transcriptome | | -l | Lineage dataset | | -o | Output name | | -c | CPU threads | | --auto-lineage | Auto-detect lineage | | --offline | Use downloaded datasets only | | --list-datasets | List available lineages |

List Available Lineages

bash
busco --list-datasets

Common Lineages

| Lineage | Use For | |---------|---------| | bacteria_odb10 | Bacteria | | archaea_odb10 | Archaea | | eukaryota_odb10 | General eukaryote | | fungi_odb10 | Fungi | | metazoa_odb10 | Animals | | vertebrata_odb10 | Vertebrates | | mammalia_odb10 | Mammals | | viridiplantae_odb10 | Plants | | saccharomycetes_odb10 | Yeasts |

Auto-Lineage Detection

bash
busco -i assembly.fasta -m genome --auto-lineage -o busco_output

Output Files

busco_output/
├── short_summary.txt           # Quick summary
├── full_table.tsv              # All BUSCO results
├── missing_busco_list.tsv      # Missing genes
└── busco_sequences/            # BUSCO gene sequences

Interpret Results

C:98.5%[S:97.0%,D:1.5%],F:0.5%,M:1.0%,n:4085

C - Complete (total)
S - Single-copy
D - Duplicated
F - Fragmented
M - Missing
n - Total BUSCO groups

Quality Thresholds

| Quality | Complete | Missing | |---------|----------|---------| | Excellent | >95% | <2% | | Good | >90% | <5% | | Acceptable | >80% | <10% | | Poor | <80% | >10% |

Complete QC Workflow

bash
#!/bin/bash
set -euo pipefail

ASSEMBLY=$1
REFERENCE=${2:-}
LINEAGE=${3:-bacteria_odb10}
OUTDIR=${4:-assembly_qc}

mkdir -p $OUTDIR

echo "=== Assembly QC ==="

# QUAST
echo "Running QUAST..."
if [ -n "$REFERENCE" ]; then
    quast.py $ASSEMBLY -r $REFERENCE -o ${OUTDIR}/quast -t 8
else
    quast.py $ASSEMBLY -o ${OUTDIR}/quast -t 8
fi

# BUSCO
echo "Running BUSCO..."
busco -i $ASSEMBLY -m genome -l $LINEAGE -o busco_run -c 8
mv busco_run ${OUTDIR}/busco

# Summary
echo ""
echo "=== QUAST Summary ==="
cat ${OUTDIR}/quast/report.txt

echo ""
echo "=== BUSCO Summary ==="
cat ${OUTDIR}/busco/short_summary*.txt

echo ""
echo "Reports saved to $OUTDIR"

Compare Assemblies

QUAST Comparison

bash
quast.py \
    spades_assembly.fa \
    flye_assembly.fa \
    canu_assembly.fa \
    -r reference.fa \
    -l "SPAdes,Flye,Canu" \
    -o assembly_comparison

BUSCO Comparison

bash
# Run BUSCO on each assembly
for asm in spades.fa flye.fa canu.fa; do
    name=$(basename $asm .fa)
    busco -i $asm -m genome -l bacteria_odb10 -o busco_${name}
done

# Generate comparison plot
generate_plot.py -wd . busco_spades busco_flye busco_canu

Python: Parse QUAST Output

python
import pandas as pd

def parse_quast(report_tsv):
    '''Parse QUAST report.tsv file.'''
    df = pd.read_csv(report_tsv, sep='\t', index_col=0)
    return df.T

stats = parse_quast('quast_output/report.tsv')
print(f"N50: {stats['N50'].values[0]}")
print(f"Total length: {stats['Total length'].values[0]}")
print(f"# contigs: {stats['# contigs'].values[0]}")

Python: Parse BUSCO Output

python
import re

def parse_busco_summary(summary_file):
    '''Parse BUSCO short summary.'''
    with open(summary_file) as f:
        text = f.read()

    pattern = r'C:(\d+\.\d+)%\[S:(\d+\.\d+)%,D:(\d+\.\d+)%\],F:(\d+\.\d+)%,M:(\d+\.\d+)%,n:(\d+)'
    match = re.search(pattern, text)

    if match:
        return {
            'complete': float(match.group(1)),
            'single': float(match.group(2)),
            'duplicated': float(match.group(3)),
            'fragmented': float(match.group(4)),
            'missing': float(match.group(5)),
            'total': int(match.group(6))
        }
    return None

result = parse_busco_summary('busco_output/short_summary.txt')
print(f"Complete: {result['complete']}%")

MetaQUAST (Metagenomes)

bash
metaquast.py metagenome_assembly.fa -o metaquast_output -t 16

Troubleshooting

Low N50

• Check coverage depth
• Consider longer reads
• Try different assembler

Low BUSCO Completeness

• Check input read quality
• Verify correct lineage dataset
• May indicate real gene loss (compare to relatives)

High Duplication in BUSCO

• Normal for polyploids
• May indicate contamination
• Check for collapsed haplotypes

Related Skills

• short-read-assembly - SPAdes assembly
• long-read-assembly - Flye/Canu assembly
• assembly-polishing - Improve accuracy
• metagenomics - Metagenome analysis

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