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Pipeline for fully automated prediction of protein coding genes with GeneMark-ES/ET and AUGUSTUS in novel eukaryotic genomes.

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The rapidly growing number of sequenced genomes requires fully automated methods for accurate gene structure annotation. With this goal in mind, we have developed BRAKER1R1R0, a combination of GeneMark-ET R2 and AUGUSTUS R3, R4, that uses genomic and RNA-Seq data to automatically generate full gene structure annotations in novel genome.

However, the quality of RNA-Seq data that is available for annotating a novel genome is variable, and in some cases, RNA-Seq data is not available, at all.

BRAKER2 is an extension of BRAKER1 which allows for fully automated training of the gene prediction tools GeneMark-EX R14, R15, R17, F1 and AUGUSTUS from RNA-Seq and/or protein homology information, and that integrates the extrinsic evidence from RNA-Seq and protein homology information into the prediction.

In contrast to other available methods that rely on protein homology information, BRAKER2 reaches high gene prediction accuracy even in the absence of the annotation of very closely related species and in the absence of RNA-Seq data.

BRAKER3 is the latest pipeline in the BRAKER suite. It enables the usage of RNA-seq and protein data in a fully automated pipeline to train and predict highly reliable genes with GeneMark-ETP and AUGUSTUS. The result of the pipeline is the combined gene set of both gene prediction tools, which only contains genes with very high support from extrinsic evidence.

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License and Disclaimer

All source code, i.e. scripts/*.pl or scripts/*.py are under the Artistic License (see


prerequisite Obtain GeneMark-ES/ET Academic License

GeneMark-ES/ET which is one of the dependencies for BRAKER requires an individual academic license  (this is free). This can be obtained as below - Download URL - genemark_es_license.png - Downloaded filename will be in the format of gm_key_64.gz - Decompress this file with gunzip gm_key_64.gz  and move it to home directory as  a hidden file under the filename .gm_key .i.e. ~/.gm_key

Copy AUGUSTUS config to a path with read/write permissions

Make a copy of AUGUSTUS config from /opt/nesi/CS400_centos7_bdw/AUGUSTUS/3.4.0-gimkl-2022a/config to path with read/write permissions .i.e. project, nobackup,home 

Example Slurm scripts

Following example uses the .fa files provided BRAKER developers

#!/bin/bash -e

#SBATCH --account nesi12345
#SBATCH --job-name braker-test
#SBATCH --cpus-per-task 4
#SBATCH --mem 1G
#SBATCH --time 02:00:00
#SBATCH --output slurmlogs/%x.%j.out
#SBATCH --error slurmlogs/%x.%j.err

module purge
module load BRAKER/3.0.2-gimkl-2022a-Perl-5.34.1

#export the path to augustus config copied above - prerequisites
export AUGUSTUS_CONFIG_PATH=/path/to/augustus/config

srun --threads=${SLURM_CPUS_PER_TASK} --genome=genome.fa --prot_seq=proteins.fa

This will generate the output directory named braker in the current working directory with content similar to below

augustus.hints.aa              braker.gtf   genemark_evidence.gff  prothint.gff
augustus.hints.codingseq       braker.log   genemark_hintsfile.gff seed_proteins.faa
augustus.hints.gtf             cmd.log      species/
augustus.hints_iter1.aa        errors/      hintsfile.gff          uniqueSeeds.gtf
augustus.hints_iter1.codingseq evidence.gff hintsfile_iter1.gff    what-to-cite.txt
augustus.hints_iter1.gff       GeneMark-EP/ prevHints.gff 
augustus.hints_iter1.gtf       GeneMark-ES/ proteins.fa