BUSCO

Overview

BUSCO (Benchmarking Universal Single-Copy Orthologs) assesses genome assembly and annotation completeness by searching for a set of highly conserved single-copy genes expected to be present in a given lineage. It reports the fraction of these marker genes found as complete, duplicated, fragmented, or missing, providing a biologically meaningful measure of assembly quality that complements purely statistical metrics like N50. BUSCO supports genome, transcriptome, and protein-level assessments and includes lineage-specific databases spanning bacteria, archaea, fungi, plants, and animals.

Installation

mamba install -c bioconda busco

Basic Usage

Evaluate a polished bacterial assembly for gene completeness using the Enterobacterales lineage database.

busco -i medaka_output/consensus.fasta \
  -o busco_output/ \
  -m genome \
  -l enterobacterales_odb10 \
  --cpu 8

Key Parameters

Flag / option	Description
`-i`	Input sequence file (genome FASTA, transcriptome FASTA, or protein FASTA).
`-o`	Output directory name for BUSCO results.
`-m`	Assessment mode: `genome`, `transcriptome`, or `proteins`.
`-l`	Lineage dataset to use (e.g. `enterobacterales_odb10`, `mammalia_odb10`). Use `busco --list-datasets` to see all available lineages.
`--cpu`	Number of CPU threads to use.
`--auto-lineage`	Automatically detect the most appropriate lineage dataset.
`--auto-lineage-prok`	Automatic lineage selection restricted to prokaryotic datasets.
`-f`	Force overwrite of existing output directory.
`--download_path`	Path where lineage databases are stored or will be downloaded.

Expected Output

BUSCO writes results to the specified output directory:

short_summary.specific.<lineage>.<output>.txt – a concise text summary reporting the number and percentage of complete (single-copy and duplicated), fragmented, and missing BUSCOs.
full_table.tsv – a detailed table listing each BUSCO marker gene and its status (Complete, Duplicated, Fragmented, or Missing).
missing_busco_list.tsv – list of BUSCO IDs that were not found.
busco_sequences/ – directory containing the nucleotide and protein sequences of identified BUSCO genes.

A high-quality bacterial assembly typically shows >95% complete BUSCOs with very few fragmented or missing genes.