BUSCO

Assessing genome assembly and annotation completeness with Benchmarking Universal Single-Copy Orthologs

This is Modified Version of BUSCO (http://busco.ezlab.org) for inhouse use.

A. Introduction

BUSCO completeness assessment employs sets of Benchmarking Universal Single-Copy Orthologs from OrthoDB (www.orthodb.org) to provide quantitative measures of the completeness of genome assemblies, annotated gene sets, and transcriptomes in terms of expected gene content. Genes that make up the BUSCO sets for each major lineage are selected from orthologous groups with genes present as single-copy orthologs in at least 90% of the species. While allowing for rare gene duplications or losses, this establishes an evolutionary informed expectation that these genes should be found as single-copy orthologs in the genome of any newly-sequenced species.

Usage of the BUSCO software requires a working installation of Python 3, HMMER 3.1, Blast+, Augustus (genome assessment only) and EMBOSS transeq (transcriptome assessment only). BUSCO genome assembly assessment first identifies candidate regions from the genome to be assessed with tBLASTn searches using BUSCO consensus sequences. Gene structures are then predicted using Augustus with BUSCO block profiles. Finally, these predicted genes, or all genes from an annotated gene set or transcriptome, are assessed using HMMER and lineage- specific BUSCO profiles to classify matc

setup

The BUSCO distribution is released as a compressed archive file (BUSCO_v1.0.tar.gz) for download.

Extracting the files to your current directory "tar -zxvf BUSCO_v1.0.tar.gz will" create the directory BUSCO, containing the required files.

Depending on the species you wish to assess, you should now download the appropriate lineage- specific profile libraries: Metazoa (M), Eukaryota (E), Arthropoda (A), Vertebrata (V), Fungi (F), or Bacteria (B) from http://busco.ezlab.org to your BUSCO directory.

Before you begin, you will need to make sure that the following required software (some only required for genome or transcriptome assessments) are installed and accessible from the command-line, e.g. set environment variable PATH=$PATH:/path/to/software/bin

• Python 3 Found in most linux package repositories

• NCBI BLAST+ http://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/LATEST/

• HMMER (HMMER 3.1b2) http://hmmer.janelia.org/

• Augustus 3.0.x (genome only) http://bioinf.uni-greifswald.de/augustus/

Make sure that the environmental variable AUGUSTUS_CONFIG_PATH was set during installion (e.g: export AUGUSTUS_CONFIG_PATH=/my_path_to_AUGUSTUS/augustus/config/)

Write access to the Augustus directory is necessary for retraining the gene predictor!

• EMBOSS tools 6.x.x (transcriptome only) ftp://emboss.open-bio.org/pub/EMBOSS/

Please check that you have the correct version of the software above.

Running BUSCO

Before starting be sure that the necessary software is installed and accessible from the command-line. This can be accomplished by adding the necessary paths to your environmental variables (e.g. PATH=$PATH:/path/to/software/bin)

One last thing to do before running BUSCO

Choose and download library of lineage-specific BUSCO data (http://busco.ezlab.org), we recommend using the largest library possible for your species. Decompress it (e.g. tar -zxvf library.tar.gz) and make sure this directory accessible (i.e. move or symlink) from the directory where the analysis is being run.

1- Genome assembly assessment:

python BUSCO_v1.1b.py -o NAME -in ASSEMBLY -l LINEAGE –m genome

NAME name to use for the run and all temporary files ASSEMBLY genome assembly file in fasta format LINEAGE path to the lineage to be used (-l /path/to/lineage)

2. Gene set assessment:

python BUSCO_v1.1b.py -o NAME -in GENE_SET -l LINEAGE -m OGS

NAME name to use for the run and temporary files GENE_SET gene set protein sequence file in fasta format LINEAGE path to the lineage to be used (-l /path/to/lineage)

3. Transcriptome assessment:

python BUSCO_v1.1b.py -o NAME -in TRANSCRIPTOME -l LINEAGE -m trans

NAME name to use for the run and temporary files TRANSCRIPTOME transcript set sequence file in fasta format LINEAGE path to the lineage to be used (-l /path/to/lineage)

options:

1. Mandatory arguments

-o name	Name used for naming output files

-in input_file	Genome assembly / gene set / transcriptome in fasta format

-l lineage	Path to the BUSCO lineage data to be used 
Example: -l /path/to/arthropoda

-m mode	Mode of analysis 
Valid options: genome, ogs, trans
Default: genome

2. Optional arguments

-h –help	Print help

-c integer	Number of CPU threads to be used
Default: 1

-sp species	Select from the pre-computed Augustus metaparameters
Selecting a closely-related species usually produces better results
Valid options: see Augustus help for list of options
Default: generic

-e evalue	Use a custom blast e-value cutoff
Default: 0.01

-f	Force overwriting of results files from a previous run with the same name

--flank N	Custom flanking genomic regions in base pairs (bp)
Used when extending selected candidate regions before gene prediction
Default: Automatically calculated flank sizes based on genome size

--long	Performs full optimization for Augustus gene finding training
Default: Off

Output

Successful execution of the BUSCO assessment pipeline will create a directory named name_OUTPUT where ‘name’ is your assigned name for the assessment run. The directory will contain several files and directories:

1. Files

short_summary_ Contains summary results in BUSCO notation and a brief breakdown of the metrics

full_table_ Complete results in tabular format with coordinates, scores and lengths of BUSCO matches

training_set_ Set of complete BUSCO matches used for training Augustus Only created during genome assessment

_tblastn Results in tabular format of tBLASTn searches with BUSCO consensus sequences

2. Directories

augustus_ Augustus-predicted genes Only created during genome assessment

augutus_proteins Corresponding Augustus-predicted proteins Only created during genome assessment

Selected Complete BUSCO matches, used for training Augustus

gb Complete BUSCO matches, GenBank format

gffs Complete BUSCO matches, GFF format

hmmer_output Tabular format HMMER output of searches with BUSCO HMMs

test with sample data

Sample data are provided to test your BUSCO setup. Execute the following commands and compare the final output ‘run_SAMPLE’ with the provided files in ‘run_TEST’.

1. Change directory to ‘sample_data’

cd sample_data/

2. Run BUSCO assessment on sequence file ‘target.fa’ in genome mode.

python BUSCO_mod.py -in target.fa -o SAMPLE -l example -m genome

3. Compare the final output ‘run_SAMPLE’ with the provided files in ‘run_TEST’.

Citation

BUSCO: assessing genome assembly and annotation completeness with single-copy orthologs. Felipe A. Simão, Robert M. Waterhouse, Panagiotis Ioannidis, Evgenia V. Kriventseva, and Evgeny M. Zdobnov Bioinformatics, published online June 9, 2015 | Abstract | Full Text PDF | doi: 10.1093/bioinformatics/btv351 Supplementary Online Materials: SOM