# Getting Started

BUSCO runs on the command line, using a Terminal or Console application. You should have some familiarity with the basics of working with the command line.

BUSCO is a software package that requires a number of third-party tools. To simplify installation, BUSCO is available as a Conda package and a Docker image with all dependencies pre-installed. It can also be installed manually though in this case care must be taken to ensure all dependencies are installed and configured correctly.

# Installation with Conda

Conda is an open-source package management and environment management system that simplifies the installation and management of software packages and their dependencies across diverse computing environments.

1. Ensure sure you have conda version 4.8.4 or higher. Enter conda -V to check. If necessary, update conda by entering conda update -n base conda.

2. To install BUSCO in the current environment, enter

   conda install -c conda-forge -c bioconda busco=5.8.0
   

3. Alternatively you can create a new environment with BUSCO installed

   conda create -n <your_env_name> -c conda-forge -c bioconda busco=5.8.0
   conda activate <your_env_name>
   

If conda takes a long time to solve the environment, you can try using mamba instead of conda. Mamba is a reimplementation of the conda package manager in C++.

conda install -c conda-forge mamba
mamba install -c conda-forge -c bioconda busco=5.8.0

# Installation with Docker

Docker is a tool for packaging and running applications and their dependencies in isolated containers, streamlining the deployment and management of software across diverse environments. Some familiarity with Docker is required to use the BUSCO Docker image. See the Docker userguide for details.

docker pull ezlabgva/busco:v5.8.0_cv1
docker run -u $(id -u) -v $(pwd):/busco_wd ezlabgva/busco:v5.8.0_cv1

docker run -u $(id -u) ezlabgva/busco:v5.8.0_cv1

Use mounts (-v) to exchange files between the host filesystem and the container filesystem. In the following example your current working directory ($(pwd)) is mounted as /busco_wd in the container. This allows you to access files in the current directory from within the container and to write files to the current directory from within the container.

docker run -u $(id -u) -v $(pwd):/busco_wd ezlabgva/busco:v5.8.0_cv1

The default working directory in the container is /busco_wd. With your non-root uid, attempts to write in the container virtual filesystem will fail if it is not a mount point. You can redefine the working directory using -w to match another mounted folder as current working directory. For example, if your BUSCO input filepath on your file system is /home/name/genome.fna

docker run -u $(id -u) -v /home/name/:/busco_wd ezlabgva/busco:v5.8.0_cv1 busco -i genome.fna

is equivalent to

docker run -u $(id -u) -v /home/name:/host_mount -w /host_mount ezlabgva/busco:v5.8.0_cv1 busco -i genome.fna

# Manual installation

To do a manual installation you will need to install the BUSCO code and all of its third-party dependencies separately. This is not recommended unless you have a specific reason to do so.

# Installing BUSCO code

The following steps should be performed in a dedicated environment. See here for more on virtual environments.

1. Clone the repository.

   git clone https://gitlab.com/ezlab/busco.git
   cd busco/
   

2. Install using pip from within the cloned directory.

   python -m pip install .
   

   NB: We recommend using a virtual environment to avoid conflicts with other Python packages.

# Third-party components

A full installation of BUSCO requires

• Python 3.3+ (2.7 is not supported from v4 onwards)
• BioPython
• Pandas
• BBMap for genome assembly statistics such as N50
• tBLASTn 2.2+ for eukaryotic genome and prokaryotic transcriptome modes
• Augustus 3.2+ for eukaryotic genome mode
• Metaeuk for eukaryotic genome and eukaryotic transcriptome modes
• Prodigal for prokaryotic genome mode
• HMMER3.1+ for all modes
• SEPP for the auto-select lineage pipeline
• R and ggplot2 for the plotting companion script

# Augustus

Augustus uses several executables and PERL scripts. Please refer to Augustus documentation for PERL requirements.

Augustus requires environment variables to be declared as follows:

export PATH="/path/to/AUGUSTUS/augustus-3.2.3/bin:$PATH"
export PATH="/path/to/AUGUSTUS/augustus-3.2.3/scripts:$PATH"
export AUGUSTUS_CONFIG_PATH="/path/to/AUGUSTUS/augustus-3.2.3/config/"

# Known bugs and unsupported versions

tBLASTn 2.4+

During development, we recognised an issue with tBLASTn versions 2.4-2.10.0 when using more than one CPU. NCBI issued a fix for the problem in version 2.10.1+. Make sure you have at least version 2.10.1+ installed.

# Supported OS

BUSCO is being developed and tested on multiple distributions of Linux (e.g. Arch Linux, CentOS, Ubuntu). We do not support macOS but BUSCO should work on it, although Augustus seems to cause troubles on some BSD-derived systems, including macOS. Consider the Docker container if you work on incompatible environments.

# Running BUSCO

# Command Line Options

# Mandatory parameters

busco -i [SEQUENCE_FILE] -m [MODE] [OTHER OPTIONS]

-i or --in defines the input file to analyse which is either a nucleotide fasta file or a protein fasta file, depending on the BUSCO mode. As of v5.1.0 the input argument can now also be a directory containing fasta files to run in batch mode.

-m or --mode sets the assessment MODE: genome, proteins, transcriptome

# Recommended parameters

-l or --lineage_dataset Specify the name of the BUSCO lineage dataset to be used, e.g. hymenoptera_odb10. A full list of available datasets can be viewed by entering busco --list-datasets. You should always select the dataset that is most closely related to the assembly or gene set you are assessing. If you are unsure, you can use the --auto-lineage option to automatically select the most appropriate dataset. BUSCO will automatically download the requested dataset if it is not already present in the download folder. You can optionally provide a path to a local dataset instead of a name, e.g. -l /path/to/my/dataset.

-c or --cpu Specify the number of threads/cores to use. Unless this is specified BUSCO will only use one CPU, which could cause a long run time.

-o or --out Give your analysis run a recognisable short name. Output folders and files will be labelled with this name. If not specified the output will take the form "BUSCO_<input_filename>"

# Optional parameters

--augustus Use augustus gene predictor for eukaryote runs.

--augustus_parameters "--PARAM1=VALUE1,--PARAM2=VALUE2" Pass additional parameters to Augustus. See Augustus documentation for parameter options. All parameters should be contained within a single string with no white space, with each parameter separated by a comma.

--augustus_species AUGUSTUS_SPECIES Specify a species for Augustus training.

--auto-lineage Run auto-lineage pipeline to find optimum lineage path.

--auto-lineage-euk Run auto-placement just on eukaryota tree to find optimal lineage path.

--auto-lineage-prok Run auto-lineage just on prokaryota trees to find optimum lineage path.

--config /path/to/config.ini Provide a config file as an alternative to command line parameters.

--contig_break n Number of contiguous Ns to signify a break between contigs in BBTools analysis. Default is n=10.

--download [dataset ...] Download dataset. Possible values are a specific dataset name, "all", "prokaryota", "eukaryota", or "virus". If used together with other command line arguments, make sure to place this last.

--download_base_url DOWNLOAD_BASE_URL Set the url to the remote BUSCO dataset location.

--download_path DOWNLOAD_PATH Specify local filepath for storing BUSCO dataset downloads. The default is a busco_downloads subdirectory in the current working directory.

-e N, --evalue N E-value cutoff for BLAST searches. Allowed formats, 0.001 or 1e-03 (Default: 1e-03).

-f, --force Force rewriting of existing files. Must be used when output files with the provided name already exist.

-h, --help Show this help message and exit.

--limit N How many candidate regions (contig or transcript) from the BLAST output to consider per BUSCO (default: 3). This option is only effective in pipelines using BLAST, i.e. the --augustus genome pipeline and the prokaryota transcriptome pipeline.

--list-datasets Print the list of available BUSCO datasets.

--long Optimize Augustus self-training mode. This adds considerably to the run time, but can improve results for some non-model organisms.

--metaeuk Use Metaeuk gene predictor.

--metaeuk_parameters "--PARAM1=VALUE1,--PARAM2=VALUE2" Pass additional arguments to Metaeuk for the first run. See Metaeuk documentation for parameter options. All parameters should be contained within a single string with no white space, with each parameter separated by a comma, e.g. --metaeuk_parameters="--max-overlap=15,--min-exon-aa=15"

--metaeuk_rerun_parameters "--PARAM1=VALUE1,--PARAM2=VALUE2" Pass additional parameters to Metaeuk for the second run. See Metaeuk documentation for parameter options. All parameters should be contained within a single string with no white space, with each parameter separated by a comma.

--miniprot Use Miniprot gene predictor (default for eukaryota in genome mode).

--offline In offline mode BUSCO will not attempt to download files. Ensure all required dataset files are already downloaded and available.

--opt-out-run-stats Opt out of data collection (from v5.6.0). Collected data is used to improve BUSCO. All collected data is anonymised and includes the pipelines used, the datasets selected, options used and runtime statistics.

--out_path OUTPUT_PATH Optional location for results folder, excluding results folder name. The default is the current working directory.

-q, --quiet Disable the info logs, displays only errors.

-r, --restart Continue a run that had already partially completed. Restarting skips calls to tools that have completed but performs all pre- and post-processing steps.

--scaffold_composition Writes ACGTN content per scaffold to a file scaffold_composition.txt. Used by BBTools.

--skip_bbtools Skip BBTools for assembly statistics.

--tar Compress some subdirectories with many files to save space.

-v, --version Show this version and exit.

# Editing BUSCO Run Configuration

BUSCO will try to use the dependencies available in your environment. For the Conda and Docker distributions these are pre-installed. For the manual installation, it is up to the user to ensure the dependencies are available. An alternative to making the tool commands available in your local environment PATH is to point to the locations of the tools in a BUSCO configuration file, config.ini. A config file template is available here. This can also be used to override the PATH if you have a reason to use a specific version of a third-party tool. All command line options can also be specified in the config file instead of on the command line. BUSCO will then first attempt to find the tools specified in the config file before falling back on the environment PATH variable. Providing input parameters through the command line will override those defined in config.ini.

To activate this config file, either pass it as a command line option

busco --config /path/to/myconfig.ini

or set the environment variable BUSCO_CONFIG_FILE with the path to the file, as follows:

export BUSCO_CONFIG_FILE="/path/to/myconfig.ini"

# Tips for running BUSCO

• Generally the lineage to select for your assessments should be the most specific lineage available, e.g. for assessing fish data one would select the actinopterygii lineage rather than the metazoa lineage.

• If you are assessing a large number of species/strains/versions etc. then to minimise runtime (at the expense of resolution) one might select a less specific lineage set with fewer BUSCOs, e.g. for assessing 20 bird genomes each with a couple of different assembly versions one might select the vertebrata or the metazoa lineages rather than the aves lineage, at least for the initial rounds of assessments.
• Assessments generally produce several folders with lots of files (see BUSCO pipelines). These are for your benefit, so that you can examine individual cases in more detail and/or use the data for downstream analyses.
• Please do take some time to check the log files, these are there for your benefit in order to highlight potential problems that may have occurred during your BUSCO run.
• Compare the results from assessing your data with like-for-like assessments of corresponding publicly available data for other closely-related species. In this way, the BUSCO results can be used to claim that your dataset is as good as, or better than, existing publicly available datasets for similar species.
• If manual curation of annotated gene sets was performed, report BUSCO results before and after curation to quantify improvements.

# Lineage datasets

BUSCO uses a set of evolutionarily-informed expectations of gene content of near-universal single-copy orthologs to assess the completeness of genome assemblies, gene sets, and transcriptomes. These expectations are derived from the analysis of a large number of evolutionarily diverse species, and are provided in the form of lineage-specific datasets. Marker genes are selected as those that are present in at least 90% of the species in a given lineage, and present in a single copy in 90% of those species. The marker genes are used to assess the completeness of the genome assembly or gene set being analysed. BUSCO scores are reported as the percentage of these marker genes that are found in the assembly or gene set.

adapted from Waterhouse et al. (2010)

BUSCO provides a set of pre-computed datasets for a wide range of organisms. Any use of these datasets for analyses in a publication or product must include the citation of the corresponding paper: https://doi.org/10.1093/molbev/msab199.

Specify a lineage dataset with the -l or --lineage_dataset option, for example

busco ... -l enterobacterales_odb10 ...

BUSCO will automatically download the requested dataset if it is not already present in the download folder. You can optionally provide a path to a local dataset instead of a name, e.g. -l /path/to/my/dataset. To print the full list of available datasets enter busco --list-datasets

For metagenomic analysis it is possible to let BUSCO selected automatically the datasets that is best suited to the input assembly, using the --auto-lineage, --auto-lineage-prok, or --auto-lineage-euk options.

The datasets contain the following directory structure:

eukaryota_odb10
├── ancestral
├── ancestral_variants
├── dataset.cfg
├── hmms
├── info
│   ├── ogs.id.info
│   └── species.info
├── lengths_cutoff
├── links_to_ODB10.txt
├── prfl
├── refseq_db.faa
├── refseq_db.faa.gz.md5
└── scores_cutoff

• The hmms directory contains the HMM file for each BUSCO.
• The info directory contains files with lists of species, genes, ortho-groups, and annotations.
• The prfl directory contains the block profile file for each BUSCO, used in the Augustus pipeline.
• The ancestral directory contains the FASTA file, consensus ancestral sequences for each BUSCO, used by BLAST in the Augustus pipeline and the prokaryotic transcriptome pipeline.
• The ancestral_variants directory contains the FASTA file, consensus & variant sequences for each BUSCO.
• The dataset.cfg file contains configuration data used in the various pipelines.
• The lengths_cutoff file contains length cut-offs for complete BUSCO matches.
• The scores_cutoff file contains score cut-offs for orthologous BUSCO matches.
• The links_to_ODB10.txt file contains annotations and links to OrthoDB for each gene.
• The refseq_db.faa file contains the reference protein sequences for each BUSCO used by Miniprot and Metaeuk.
• The missing_in_parasitic.txt file, where present, contains a list of genes present in lineages containing clades with reduced parasitic genomes (e.g. fungi_odb10).

# Download and automated update

BUSCO can obtain the last version of the lineage datasets. If the name of a dataset is passed, e.g. -l bacteria_odb10, BUSCO will download it automatically. If a full path is given to BUSCO using -l /my/own/path/bacteria_odb10, this automated management will be disabled. Otherwise, files used during the automated lineage selection process are also automatically obtained by BUSCO.

By default, if a new version of a file is available, BUSCO will warn you. If you pass the argument --update-data, busco will replace the current file or folder with the up-to-date version and archive the previous one.

Individual datasets can also be downloaded using the busco --download command, e.g. busco --download bacteria_odb10. It is also possible to do a bulk download by using the following arguments in place of the dataset name: "all", "prokaryota", "eukaryota", or "virus". Please exercise restraint when using the "download" command - excessive use of this command will put a strain on the BUSCO server and may result in your IP address being blocked.

# Offline

If you are running BUSCO in an environment that does not have access to the Internet, you can pass the --offline argument to prevent BUSCO from attempting any download. Make sure you have first downloaded all the datasets you will need using the busco --download command. The location of the download folder should then be passed using the command line flag: --download_path /path/to/datasets.

A valid download folder looks as follows:

busco_downloads/
├── file_versions.tsv
├── information
│   └── lineages_list.2021-12-14.txt
├── lineages
│   ├── agaricales_odb10
│   ├── agaricomycetes_odb10
│   ├── archaea_odb10
│   ├── ascomycota_odb10
│   ├── bacillales_odb10
│   ├── bacteria_odb10
│   ├── endopterygota_odb10
│   ├── eudicots_odb10
│   ├── eukaryota_odb10
│   ├── fungi_odb10
│   ├── hemiptera_odb10
│   ├── insecta_odb10
│   ├── mammalia_odb10
│   ├── metazoa_odb10
│   ├── methanococcales_odb10
│   ├── natrialbales_odb10
│   ├── primates_odb10
│   ├── saccharomycetes_odb10
│   └── solanales_odb10
└── placement_files
    ├── list_of_reference_markers.archaea_odb10.2019-12-16.txt
    ├── mapping_taxid-lineage.archaea_odb10.2019-12-16.txt
    ├── mapping_taxids-busco_dataset_name.archaea_odb10.2019-12-16.txt
    ├── supermatrix.aln.archaea_odb10.2019-12-16.faa
    ├── tree.archaea_odb10.2019-12-16.nwk
    ├── tree_metadata.archaea_odb10.2019-12-16.tx

# BUSCO Pipelines

The BUSCO output folder name is BUSCO_<input_filename> by default, but this can be changed by using the -o or --out option. This directory will contain several files and directories, which will vary depending on which pipeline you are using.

# Genome mode

The genome mode is used to assess the completeness of genome assemblies. It uses the --mode option set to genome. The input file should be a nucleotide fasta file. There are distinct pipelines for eukaryotic and prokaryotic genomes. BUSCO decides which pipeline to use depending on the dataset selected. Each pipeline uses a different gene predictor tool, explained below. The predicted genes are then passed to HMMER, which scores them against the HMM profiles for the BUSCO marker genes in a given dataset.

The HMMER results are then passed through a number of filters to categorise the gene matches as Complete and Single-copy, Complete and Duplicated, Fragmented or Missing. These filters ensure that:

• no BUSCO IDs is categorised multiple times
• no gene match is assigned to multiple BUSCO IDs
• matches in which the top match is significantly better than other matches are treated as Single-copy matches
• no two gene matches overlap. If they do, the match with the lower score is discarded. BUSCO will first examine the individual exons of a gene match to see if each exon is matched to the HMM profile. If an exon at the flank of a gene match is not matched to the HMM profile by HMMER, and removing this exon would resolve the overlap, BUSCO will discard the exons and update the gene coordinates accordingly. This can lead to the coordinates reported in the full_table.tsv file being different from those in the GFF file.

Note: When running BUSCO using the fungi_odb10 dataset, BUSCO will recognise if the missing BUSCO marker genes match the pattern of those in parasitic-reduced genomes. This will be reported along with a recalculated score that would be obtained by disregarding these missing genes.

# Eukaryota

As of v5.7.0, Miniprot is the default tool for eukaryotic genome mode. Miniprot is not a gene predictor, but a gene mapper, and uses a reference protein database (provided in the BUSCO datasets) to map proteins to the genome. Miniprot is generally faster than Augustus and Metaeuk (except on fungi, in which Metaeuk is still faster), and typically yields slightly higher completion scores. However, Miniprot may underperform for highly divergent assemblies (with respect to the species in the BUSCO datasets), due to its limited sensitivity to divergent orthologs. Caution should be exercised with the reported gene predictions, as many of these may contain internal stop codons and may not be suitable for further analysis. BUSCO will report anomalies detected in the final results.

# Miniprot pipeline (default for eukaryota)

The typical output of the Miniprot pipeline is as follows:

<output_folder>
├── logs
│   ├── *_err.log
│   ├── *_out.log
├── run_<dataset>
│   ├── busco_sequences
│   │   ├── fragmented_busco_sequences
│   │   ├── multi_copy_busco_sequences
│   │   └── single_copy_busco_sequences
│   ├── full_table.tsv
│   ├── hmmer_output
│   ├── miniprot_output
│   │   ├── ref.mpi
│   │   └── translated_proteins
│   ├── missing_busco_list.tsv
│   ├── short_summary.json
│   └── short_summary.txt
├── short_summary.specific.<dataset>.<output_folder>.json
└── short_summary.specific.<dataset>.<output_folder>.txt

• The logs/ folder contains a detailed busco.log file (with DEBUG notifications) and the captured stderr and stdout streams of each third party software.
• The busco_sequences/ subdirectory contains protein sequence files in FASTA format (*.faa) and GFF files (*.gff) for each BUSCO gene identified. Nucleotide coding sequences are not provided in the Miniprot pipeline.
• The full_table.tsv file contains the complete results in a tabular format with scores and lengths of BUSCO matches, and coordinates (for genome mode) or gene/protein IDs (for transcriptome or protein modes).
• The missing_busco_list.tsv file contains a list of missing BUSCOs.
• The short_summary.*.txt and short_summary.*.json files contain a plain text and JSON summary of the results.
• The hmmer_output directory contains tabular format HMMER output of searches with BUSCO HMMs.

# Metaeuk pipeline

Metaeuk was introduced as the default gene predictor in v5.0.0, replacing Augustus. To use the Metaeuk pipeline add the --metaeuk option on the command line or set use_metaeuk=True in the config file. Designed for eukaryotic metagenomes, Metaeuk is a fast and accurate gene predictor that uses a reference protein database (provided in the BUSCO datasets) to predict genes. Metaeuk is faster than Augustus. It also uses less memory than Miniprot.

The Metaeuk pipeline performs an initial run and then a rerun with different parameters in an attempt to find matches for BUSCOs that were Missing or Fragmented in the first pass. The directory structure is slightly different as a result:

<output_folder>
├── logs
├── run_<dataset>
│   ├── busco_sequences
│   │   ├── fragmented_busco_sequences
│   │   ├── multi_copy_busco_sequences
│   │   └── single_copy_busco_sequences
│   ├── full_table.tsv
│   ├── hmmer_output
│   │   ├── initial_run_results
│   │   └── rerun_results
│   ├── metaeuk_output
│   │   ├── combined_nucl_seqs.fas
│   │   ├── combined_pred_proteins.fas
│   │   ├── initial_results
│   │   └── rerun_results
│   ├── missing_busco_list.tsv
│   ├── short_summary.json
│   └── short_summary.txt
├── short_summary.specific.<dataset>.<output_folder>.json
└── short_summary.specific.<dataset>.<output_folder>.txt

• For the Metaeuk pipeline, the busco_sequences/ subdirectory also contains nucleotide coding sequence files in FASTA format (*.fna).

# Augustus pipeline

Augustus is a widely used gene predictor for eukaryotic genomes. It is the default gene predictor for eukaryotic genomes in BUSCO v4.0.0 and earlier. To use the Augustus pipeline add the --augustus option on the command line or set use_augustus=True in the config file. Augustus is a self-training gene predictor, and requires a reference file for training. BUSCO specifies a default training set for each dataset lineage, but it is possible to specify an alternative training set using the --augustus_species option. Augustus gene predictions are the most reliable for downstream analysis.

The Augustus pipeline includes a number of Augustus scripts. The output directory structure is built around the various steps in this procedure. BUSCO also performs a second pass to find matches for BUSCOs that were Missing or Fragmented in the first pass. The rerun parameters are obtained by training Augustus on the results found in the first pass.

<output_folder>
├── blast_db
├── logs
├── run_<dataset>
│   ├── augustus_output
│   │   ├── extracted_proteins
│   │   ├── gb
│   │   ├── gff
│   │   ├── predicted_genes_initial_run
│   │   ├── predicted_genes_rerun
│   │   ├── retraining_parameters
│   │   │   └── BUSCO_<output_folder>
│   │   └── training_set.db
│   ├── blast_output
│   │   ├── ancestral_variants_missing_and_frag_rerun
│   │   ├── coordinates.tsv
│   │   ├── coordinates_missing_and_frag_rerun.tsv
│   │   ├── sequences
│   │   ├── tblastn.tsv
│   │   └── tblastn_missing_and_frag_rerun.tsv
│   ├── busco_sequences
│   │   ├── fragmented_busco_sequences
│   │   ├── multi_copy_busco_sequences
│   │   └── single_copy_busco_sequences
│   ├── full_table.tsv
│   ├── hmmer_output
│   │   ├── initial_run_results
│   │   └── rerun_results
│   ├── missing_busco_list.tsv
│   ├── short_summary.json
│   └── short_summary.txt
├── short_summary.specific.<dataset>.<output_folder>.json
└── short_summary.specific.<dataset>.<output_folder>.txt

• The training parameters found in the retraining_parameters subdirectory are used in the Augustus rerun, and can also be used with Augustus for subsequent analysis if so desired.
• The blast_db directory contains the BLAST database used in the Augustus pipeline. This is independent of the dataset used, so it is stored one level higher than the dataset-specific results.

# Prokaryota

For prokaryotic genomes Prodigal is the default gene predictor. Prodigal predicts genes ab-initio, without any reference file. Prodigal also allows the translation table to be specified, which is useful for annotating genomes with non-standard genetic codes. BUSCO datasets for prokaryota already specify the optimal genetic code for a given clade. In cases in which multiple genetic codes are present within a single clade, BUSCO will automatically assess which translation table yields the highest coding density.

The typical output of the Prodigal pipeline is as follows:

<output_folder>
├── logs
├── prodigal_output
│   └── predicted_genes
│       └── tmp
├── run_<dataset>
│   ├── busco_sequences
│   │   ├── fragmented_busco_sequences
│   │   ├── multi_copy_busco_sequences
│   │   └── single_copy_busco_sequences
│   ├── full_table.tsv
│   ├── hmmer_output
│   ├── missing_busco_list.tsv
│   ├── short_summary.json
│   └── short_summary.txt
├── short_summary.specific.<dataset>.<output_folder>.json
└── short_summary.specific.<dataset>.<output_folder>.txt

• In cases in which Prodigal is run multiple times using different genetic codes the output of each run can be found in the prodigal_output>predicted_genes>tmp.

# Transcriptome mode

Transcriptome mode can be selected using the --mode option set to transcriptome. Depending on the dataset used, BUSCO will run either the eukaryota pipeline or the prokaryota pipeline.

# Eukaryota

For eukaryotic transcriptomes BUSCO uses the Eukaryota Metaeuk pipeline, and has the same output directory structure. The input file should be a nucleotide fasta file.

# Prokaryota

For prokaryotic transcriptome mode, BUSCO runs BLAST searches to find matches for the BUSCO marker genes, followed by HMMER scoring of all six frame translations. These translations are naive, ignoring start and stop codons only in order to apply hmmsearch and do not represent proteins The input file should be a nucleotide fasta file. The typical output of the prokaryotic transcriptome pipeline is as follows:

<output_folder>
├── blast_db
├── logs
├── run_<dataset>
│   ├── blast_output
│   │   ├── coordinates.tsv
│   │   ├── sequences
│   │   └── tblastn.tsv
│   ├── busco_sequences
│   │   ├── fragmented_busco_sequences
│   │   ├── multi_copy_busco_sequences
│   │   └── single_copy_busco_sequences
│   ├── full_table.tsv
│   ├── hmmer_output
│   ├── missing_busco_list.tsv
│   ├── short_summary.json
│   └── short_summary.txt
├── short_summary.specific.<dataset>.<output_folder>.json
├── short_summary.specific.<dataset>.<output_folder>.txt
└── translated_proteins

# Protein mode

Protein mode can be selected using the --mode option set to proteins. The input file should be a protein fasta file. The input file is passed directly to HMMER, which scores the proteins against the HMM profiles for the BUSCO marker genes in a given dataset.

The typical output of the protein pipeline is as follows:

<output_folder>
├── logs
│   ├── busco.log
│   ├── hmmsearch_err.log
│   └── hmmsearch_out.log
├── run_<dataset>
│   ├── busco_sequences
│   │   ├── fragmented_busco_sequences
│   │   ├── multi_copy_busco_sequences
│   │   └── single_copy_busco_sequences
│   ├── full_table.tsv
│   ├── hmmer_output
│   │   ├── initial_run_results
│   │   └── rerun_results
│   ├── missing_busco_list.tsv
│   ├── short_summary.json
│   └── short_summary.txt
├── short_summary.specific.<dataset>.<output_folder>.json
└── short_summary.specific.<dataset>.<output_folder>.txt

# Auto-select lineage

# Automated lineage selection

Since v4.0.0, it is possible to let BUSCO automatically select the dataset that is best suited to the input assembly. This is particularly useful for metagenomic analysis, where the lineage of the input assembly may be unknown. The Auto-Select Lineage pipeline can be invoked using the --auto-lineage option, e.g.

busco -m MODE -i INPUT -o OUTPUT --auto-lineage

This pipeline runs the respective BUSCO pipelines on the generic lineage datasets for the domains archaea, bacteria and eukaryota. The --auto-lineage-euk and --auto-lineage-prok options run auto-placement just on eukaryota and prokaryota (archaea, bacteria) parent datasets, respectively, and may save on computational resources if compatible with your experimental goal. BUSCO selects the phylogenetic tree associated with the highest scoring parent dataset. BUSCO then uses SEPP to place the input assembly on the selected tree. The most appropriate BUSCO dataset is selected based on this placement.

BUSCO evaluations are valid when an appropriate dataset is used, i.e., the dataset belongs to the lineage of the species being analysed. Because of overlapping markers/spurious matches among domains, BUSCO matches in another domain do not necessarily mean that your genome/proteome contains sequences from this domain. However, a high BUSCO score in multiple domains might help you identify possible contaminations.

The typical output directory structure for the auto-select lineage pipeline is as follows:

<output_folder>
├── auto_lineage
│   ├── run_archaea_odb10
│   │   ├── busco_sequences
│   │   │   ├── fragmented_busco_sequences
│   │   │   ├── multi_copy_busco_sequences
│   │   │   └── single_copy_busco_sequences
│   │   ├── full_table.tsv
│   │   ├── hmmer_output
│   │   ├── missing_busco_list.tsv
│   │   ├── placement_files
│   │   │   ├── marker_genes.fasta
│   │   │   ├── output_alignment.fasta
│   │   │   ├── output_alignment_masked.fasta
│   │   │   ├── output_placement.json
│   │   │   └── output_rename-json.py
│   │   ├── short_summary.json
│   │   └── short_summary.txt
│   ├── run_bacteria_odb10
│   │   ├── busco_sequences
│   │   │   ├── fragmented_busco_sequences
│   │   │   ├── multi_copy_busco_sequences
│   │   │   └── single_copy_busco_sequences
│   │   ├── full_table.tsv
│   │   ├── hmmer_output
│   │   ├── missing_busco_list.tsv
│   │   ├── short_summary.json
│   │   └── short_summary.txt
│   └── run_eukaryota_odb10
│       ├── busco_sequences
│       │   ├── fragmented_busco_sequences
│       │   ├── multi_copy_busco_sequences
│       │   └── single_copy_busco_sequences
│       ├── full_table.tsv
│       ├── hmmer_output
│       ├── miniprot_output
│       │   ├── ref.mpi
│       │   └── translated_proteins
│       ├── missing_busco_list.tsv
│       ├── short_summary.json
│       └── short_summary.txt
├── logs
├── prodigal_output
│   └── predicted_genes
│       └── tmp
├── run_archaea_odb10 -> auto_lineage/run_archaea_odb10
├── run_methanococcales_odb10
│   ├── busco_sequences
│   │   ├── fragmented_busco_sequences
│   │   ├── multi_copy_busco_sequences
│   │   └── single_copy_busco_sequences
│   ├── full_table.tsv
│   ├── hmmer_output
│   ├── missing_busco_list.tsv
│   ├── short_summary.json
│   └── short_summary.txt
├── short_summary.generic.archaea_odb10.<output_folder>.json
├── short_summary.generic.archaea_odb10.<output_folder>.txt
├── short_summary.specific.methanococcales_odb10.<output_folder>.json
└── short_summary.specific.methanococcales_odb10.<output_folder>.txt

• The auto-lineage folder contains the results for the three parent datasets, archaea_odb10, bacteria_odb10, and eukaryota_odb10.
• The prodigal_output directory is located at the top level, as these results are used for both archaea_odb10 and bacteria_odb10 runs, along with any other possible subsequent prokaryotic runs.
• The run_<dataset> folders contain the results for the respective parent datasets. The final selected dataset will be at the top level of the results folder.
• In the event that placement cannot be performed due to insufficient marker genes, BUSCO will report the results from the optimal parent dataset. This results folder will be soft-linked from the top level of the results folder.
• The short_summary* files for the parent dataset ("generic") and the final selected dataset ("specific") are located at the top level of the results folder.

# Batch mode

BUSCO allows you to run multiple analyses in batch mode. The input is a directory containing multiple fasta files. The output is a directory containing subdirectories named after the input files, which otherwise contain the same structure as described above.

<output_folder>
├── GCF_000091665.1_ASM9166v1_genomic.fna
│   ├── logs
│   │   ├── bbtools_err.log
│   │   ├── bbtools_out.log
│   │   ├── hmmsearch_err.log
│   │   ├── hmmsearch_out.log
│   │   ├── prodigal_err.log
│   │   └── prodigal_out.log
│   ├── prodigal_output
│   │   └── predicted_genes
│   ├── run_bacteria_odb10
│   │   ├── busco_sequences
│   │   ├── full_table.tsv
│   │   ├── hmmer_output
│   │   ├── missing_busco_list.tsv
│   │   ├── short_summary.json
│   │   └── short_summary.txt
│   ├── short_summary.specific.bacteria_odb10.GCF_000091665.1_ASM9166v1_genomic.fna.json
│   └── short_summary.specific.bacteria_odb10.GCF_000091665.1_ASM9166v1_genomic.fna.txt
├── GCF_000226975.2_ASM22697v3_genomic.fna
│   ├── logs
│   │   ├── bbtools_err.log
│   │   ├── bbtools_out.log
│   │   ├── hmmsearch_err.log
│   │   ├── hmmsearch_out.log
│   │   ├── prodigal_err.log
│   │   └── prodigal_out.log
│   ├── prodigal_output
│   │   └── predicted_genes
│   ├── run_bacteria_odb10
│   │   ├── busco_sequences
│   │   ├── full_table.tsv
│   │   ├── hmmer_output
│   │   ├── missing_busco_list.tsv
│   │   ├── short_summary.json
│   │   └── short_summary.txt
│   ├── short_summary.specific.bacteria_odb10.GCF_000226975.2_ASM22697v3_genomic.fna.json
│   └── short_summary.specific.bacteria_odb10.GCF_000226975.2_ASM22697v3_genomic.fna.txt
├── batch_summary.txt
└── logs
    └── busco.log

• The primary difference is that there is a top-level logs/ directory containing the busco.log file, which contains the logs for all the analyses in the batch. There are separate logs/ folders for each run, containing the stderr and stdout of each third party software for that run.

# Interpreting the results

BUSCO provides a quantitative assessment of the completeness in terms of expected gene content of a genome assembly, transcriptome, or annotated gene set. The results are simplified into categories of Complete and single-copy, Complete and duplicated, Fragmented, or Missing BUSCOs, where "BUSCOs" is shorthand for "BUSCO marker genes".

BUSCO completeness results make sense only in the context of the biology of your organism. You have to understand whether missing or duplicated genes are of biological or technical origin. For instance, a high level of duplication may be explained by a recent whole duplication event (biological) or a chimeric assembly of haplotypes (technical). Transcriptomes and protein sets that are not filtered for isoforms will lead to a high proportion of duplicates. Therefore, you should filter them before a BUSCO analysis. Finally, focusing on specific tissues or specific life stages and conditions in a transcriptomic experiment is unlikely to produce a BUSCO-complete transcriptome. In this case, consistency across your samples is what you will be aiming for.

If you need help or suggestions, use the BUSCO issues board to exchange with the BUSCO development team and other users.

# Complete

If found to be complete, whether single-copy or duplicated, the BUSCO matches have scored within the expected range of scores and within the expected range of length alignments to the BUSCO profile. If in fact an ortholog is not present in the input dataset, or the ortholog is only partially present (highly fragmented), and a high-identity full-length homolog is present, it is possible that this homolog could be mistakenly identified as the complete BUSCO. The score thresholds are optimised to minimise this possibility, but it can still occur.

# Fragmented

If found to be fragmented, the BUSCO matches have scored within the range of scores but not within the range of length alignments to the BUSCO profile. For transcriptomes or annotated gene sets this indicates incomplete transcripts or gene models. For genome assemblies this could indicate either that the gene is only partially present or that the sequence search and gene prediction steps failed to produce a full-length gene model even though the full gene could indeed be present in the assembly. When running eukaryotic datasets with the Metaeuk (default) or Augustus gene predictor, matches that produce such fragmented results are given a "second chance" with a second round of sequence searches and gene predictions with ancestral variants of the missing sequences and, in the case of Augustus, parameters trained on those BUSCOs that were found to be complete. However, this can still fail to recover the whole gene. Some fragmented BUSCOs from genome assembly assessments could therefore be complete but are just too divergent or have very complex gene structures, making them very hard to locate and predict in full.

# Missing

If found to be missing, there were either no significant matches at all, or the BUSCO matches scored below the range of scores for the BUSCO profile. For transcriptomes or annotated gene sets this indicates that these orthologs are indeed missing or the transcripts or gene models are so incomplete/fragmented that they could not even meet the criteria to be considered as fragmented. For genome assemblies this could indicate either that these orthologs are indeed missing, or that the sequence search step failed to identify any significant matches, or that the gene prediction step failed to produce even a partial gene model that might have been recognised as a fragmented BUSCO match. Like for fragments, when running eukaryotic datasets with the Metaeuk (default) or Augustus pipelines, BUSCOs missing after the first round are given a "second chance" with a second round of sequence searches and gene predictions with ancestral variants of the missing sequences and, in the case of Augustus, parameters trained on those BUSCOs that were found to be complete. However, this can still fail to recover the whole gene. Some missing BUSCOs from genome assembly assessments could therefore be partially present, and even possibly (but unlikely) complete, but they are just too divergent or have very complex gene structures, making them very hard to locate and predict correctly or even partially.

# Reporting BUSCO

• Report results in simple BUSCO notation: C:89.0%[S:85.8%,D:3.2%],F:6.9%,M:4.1%,n:3023
• Use the generate_plot.py (see below) script to produce simple graphical summaries (that are easily customisable) for your publication’s supporting online information.
• Report the versions you used for all third party components. We highly recommend using the BUSCO container, whose version is sufficient to safely reproduce a run.
• Report the BUSCO set(s) you used for your assessments. Mention the creation date of the dataset, not only the name, e.g. archaea_odb10 (2019-01-04).
• Report the BUSCO options you used.
• Report the version(s) of the genome assembly, annotated gene set, or transcriptome that you assessed.

# Companion scripts

# Plot

The scripts/generate_plot.py script allows users to quickly view their BUSCO summary results in an easily-understandable bar chart. The scripts/generate_plot.py uses R (https://www.r-project.org/) and ggplot2 (http://ggplot2.org/) to summarise BUSCO runs for side-by-side comparisons. The script produces a PNG image (if both R and ggplot2 are available), as well as an R source code file that can be used to run on a different machine where both R and ggplot2 are available or which can be edited to fully customise the resulting bar chart (colours, labels, fonts, axes, etc.).

usage: python3 generate_plot.py -wd [WORKING_DIRECTORY] [OTHER OPTIONS]

BUSCO plot generation tool.
Place all BUSCO short summary files (short_summary.[generic|specific].dataset.label.txt) in a single folder. It will be your working directory, in which the generated plot files will be written
See also the user guide for additional information

required arguments:
  -wd PATH, --working_directory PATH
                        Define the location of your working directory

optional arguments:
  -rt RUN_TYPE, --run_type RUN_TYPE
                        type of summary to use, `generic` or `specific`
  --no_r                To avoid to run R. It will just create the R script file in the working directory
  -q, --quiet           Disable the info logs, displays only errors
  -h, --help            Show this help message and exit

To run scripts/generate_plot.py, first create a folder, e.g. mkdir BUSCO_summaries, and then copy the BUSCO short summary file from each of the runs you want to plot into this folder.

cp XX1/short_summary.*.lineage_odb10.XX1.txt BUSCO_summaries/.
cp XX2/short_summary.*.lineage_odb10.XX2.txt BUSCO_summaries/.
cp XX3/short_summary.*.lineage_odb10.XX3.txt BUSCO_summaries/.

Then simply run the script giving as argument the name (or full path if you are not in same working directory) of the folder you created containing the summaries you wish to plot.

python3 scripts/generate_plot.py –wd BUSCO_summaries
python3 scripts/generate_plot.py –wd /full/path/to/my/folder/BUSCO_summaries

The resulting PNG image and the corresponding R source code file will be produced in the same folder containing the BUSCO summaries. By default, the run name is used as the label for each plotted result, and this is automatically extracted from the short summary file name: so for short_summary.generic.lineage_odb10.XX1.txt the label would be XX1. You can modify this as long as you keep the naming convention: short_summary.generic.lineage_odb10.[edit_name_here].txt or you can simply edit the R source code file to change any plotting parameters and produce a personalised bar chart running the code manually in your R environment.

Example scripts/generate_plot.py bar chart:

mkdir my_summaries
cp SPEC1/short_summary.generic.lineage1_odb10.SPEC1.txt my_summaries/.
cp SPEC2/short_summary.generic.lineage2_odb10.SPEC2.txt my_summaries/.
cp SPEC3/short_summary.specific.lineage2_odb10.SPEC3.txt my_summaries/.
cp SPEC4/short_summary.generic.lineage3_odb10.SPEC4.txt my_summaries/.
cp SPEC5/short_summary.generic.lineage4_odb10.SPEC5.txt my_summaries/.
python3 scripts/generate_plot.py –wd my_summaries

# Phylogenomics

The repository https://gitlab.com/ezlab/busco_usecases contains the script that was used to produce the phylogenomics portion of the BUSCO v3 (PMID: 29220515) paper. It has not been ported to BUSCO v4.

# Troubleshooting

If you need help, please search the BUSCO issues board or open a new issue if your question has not previously been answered.

# Citation

The novelties introduced in BUSCO v4 and v5 and the current BUSCO datasets (*_odb10) are described here.
The correct citation for these versions of the software and datasets is:

Mosè Manni, Matthew R Berkeley, Mathieu Seppey, Felipe A Simão, Evgeny M Zdobnov, BUSCO Update: Novel and Streamlined Workflows along with Broader and Deeper Phylogenetic Coverage for Scoring of Eukaryotic, Prokaryotic, and Viral Genomes. Molecular Biology and Evolution, Volume 38, Issue 10, October 2021, Pages 4647–4654

The following protocol covers the various BUSCO running modes and workflows, BUSCO setup, guidelines to interpret the results, and additional analyses, e.g., for building phylogenomic trees and visualizing syntenies using BUSCO results:

Manni, M., Berkeley, M. R., Seppey, M., & Zdobnov, E. M. (2021). BUSCO: Assessing genomic data quality and beyond. Current Protocols, 1, e323. doi: 10.1002/cpz1.323

# Citations of third-party tools

As BUSCO is reliant on a number of third-party tools, please also cite the following (depending on the pipeline used):

Miniprot

Li, H. (2023) Protein-to-genome alignment with miniprot. Bioinformatics, 39, btad014 [PMID: 36648328].

Metaeuk

Levy Karin E, Mirdita M and Soeding J. MetaEuk – sensitive, high-throughput gene discovery and annotation for large-scale eukaryotic metagenomics. Microbiome. 2020; 8:48

Augustus

Mario Stanke, Mark Diekhans, Robert Baertsch, David Haussler (2008). Using native and syntenically mapped cDNA alignments to improve de novo gene finding. Bioinformatics, 24(5), pages 637–644, doi: 10.1093/bioinformatics/btn013

Prodigal

Hyatt D, Chen GL, LoCascio PF, Land ML, Larimer FW, Hauser LJ. Prodigal: prokaryotic gene recognition and translation initiation site identification. BMC Bioinformatics. 2010 Mar 8;11:119. doi: 10.1186/1471-2105-11-119. PMID: 20211023; PMCID: PMC2848648.

HMMER

hmmer.org

Eddy SR. (2011) Accelerated Profile HMM Searches. PLoS Comput Biol 7(10): e1002195. doi: 10.1371/journal.pcbi.1002195

BLAST+

Camacho C, Coulouris G, Avagyan V, Ma N, Papadopoulos J, Bealer K, Madden TL. BLAST+: architecture and applications. BMC Bioinformatics. 2009 Dec 15;10:421. doi: 10.1186/1471-2105-10-421. PMID: 20003500; PMCID: PMC2803857.

SEPP

Mirarab S, Nguyen N, Warnow T. SEPP: SATé-enabled phylogenetic placement. Pac Symp Biocomput. 2012:247-58. PMID: 22174271; PMCID: PMC3243193.

# License

The BUSCO software is licensed under the MIT License.

The BUSCO datasets are licensed under the Creative Commons Attribution-NoDerivatives 4.0 International License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nd/4.0/ or send a letter to Creative Commons, PO Box 1866, Mountain View, CA 94042, USA.
Any use of these datasets for analyses in a publication or product must include the citation of the corresponding paper: https://doi.org/10.1093/molbev/msab199.