TSEBRA is a combiner tool that selects transcripts from gene predictions based on the support by extrisic evidence in form of introns and start/stop codons. It was developed to combine BRAKER1¹ and BRAKER2² predicitons to increase their accuracies.

Python 3.5.2 or higher is required.

Download TSEBRA:

git clone https://github.com/LarsGab/TSEBRA 

Or download TSEBRA as submodule of BRAKER with:

git clone --recurse-submodules https://github.com/Gaius-Augustus/BRAKER

The main script is ./bin/tsebra.py. For usage information run ./bin/tsebra.py --help.

TSEBRA needs a list of gene prediciton files, a list of hintfiles and a configuration file as input.

The gene prediction files needs to be in gtf format. This is the standard output format of a BRAKER or AUGUSTUS^3,4 gene prediciton.

Example:

2L      AUGUSTUS        gene    83268   87026   0.88    -       .       g5332
2L      AUGUSTUS        transcript      83268   87026   0.88    -       .       g5332.t1
2L      AUGUSTUS        intron  84278   87019   1       -       .       transcript_id "file_1_file_1_g5332.t1"; gene_id "file_1_file_1_g5332";
2L      AUGUSTUS        CDS     87020   87026   0.88    -       0       transcript_id "file_1_file_1_g5332.t1"; gene_id "file_1_file_1_g5332";
2L      AUGUSTUS        exon    87020   87026   .       -       .       transcript_id "file_1_file_1_g5332.t1"; gene_id "file_1_file_1_g5332";

The hint files have to be in gff format, the last column must include an attribute for the source for the hint with 'src=' and can include the number of hints supporting the gene structure segment with 'mult='. This is the standard file format of the hintfiles.gff in a BRAKER working directory.

Example:

2L      ProtHint        intron  279806  279869  2       +       .       src=P;mult=25;pri=4;al_score=0.437399;
2L      ProtHint        intron  275252  275318  2       -       .       src=P;mult=19;pri=4;al_score=0.430006;
2L      ProtHint        stop    293000  293002  1       +       0       grp=7220_0:002b08_g42;src=C;pri=4;
2L      ProtHint        intron  207632  207710  1       +       .       grp=7220_0:002afa_g26;src=C;pri=4;
2L      ProtHint        start   207512  207514  1       +       0       grp=7220_0:002afa_g26;src=C;pri=4;

The configuration file has to include three types of parameter:

1. The weight for each hint source. A weight is set to 1, if the weight for a source is not determined in the cfg file.
2. Required fraction of supported introns or supported start/stop-codons for a transcript.
3. Allowed difference between two overlapping transcripts for each feature type.

Example:

# src weights
P 0.1
E 10
C 5
M 1
# Low evidence support
intron_support 0.75
stasto_support 1
# Feature differences 
e_1 0
e_2 0.5
e_3 25
e_4 10

The recommended and most common usage for TSEBRA is to combine the resulting braker.gtf files of a BRAKER1 and a BRAKER2 run using the hintsfile.gff from both working directories. However, TSEBRA can be applied to any number (>1) of gene predictions and hint files as long as they are in the correct format.

A common case might be that a user wants to annotate a novel genome with BRAKER and has:

• a novel genome with repeats masked: genome.fasta.masked,
• hints for intron positions from RNA-seq readsrna_seq_hints.gff,
• database of homologous proteins: proteins.fa.

1. Run BRAKER1 and BRAKER2 for example with

### BRAKER1
braker.pl --genome=genome.fasta.masked --hints=rna_seq_hints.gff \ 
            --softmasking --species=species_name --workingdir=braker1_out
    
### BRAKER2
braker.pl --genome=genome.fasta.masked --prot_seq=proteins.fa \ 
    --softmasking --species=species_name --epmode --prg=ph \ 
    --workingdir=braker2_out

2. Make sure that the gene and transcript IDs of the gene prediction files are in order (this step is optional)

./bin/fix_gtf_ids.py --gtf braker1_out/braker.gtf --out braker1_fixed.gtf
./bin/fix_gtf_ids.py --gtf braker2_out/braker.gtf --out braker2_fixed.gtf

3. Combine predicitons with TSEBRA

./bin/tsebra.py -g braker1_fixed.gtf,braker2_fixed.gtf -c default.cfg \ 
    -e braker1_out/hintsfile.gff,braker2_out/hintsfile.gff \
    -o braker1+2_combined.gtf

The combined gene prediciton is braker1+2_combined.gtf.

A small example is located at example/. Run ./example/run_prevco_example.sh to execute the example and to check if TSEBRA runs properly.

All source code, i.e. bin/*.py are under the Artistic License (see https://opensource.org/licenses/Artistic-2.0).

[1] Hoff, Katharina J, Simone Lange, Alexandre Lomsadze, Mark Borodovsky, and Mario Stanke. 2015. “BRAKER1: Unsupervised Rna-Seq-Based Genome Annotation with Genemark-et and Augustus.” Bioinformatics 32 (5). Oxford University Press: 767--69.↑

[2] Tomas Bruna, Katharina J. Hoff, Alexandre Lomsadze, Mario Stanke and Mark Borodvsky. 2021. “BRAKER2: automatic eukaryotic genome annotation with GeneMark-EP+ and AUGUSTUS supported by a protein database." NAR Genomics and Bioinformatics 3(1):lqaa108.↑***

[3] Stanke, Mario, Mark Diekhans, Robert Baertsch, and David Haussler. 2008. “Using Native and Syntenically Mapped cDNA Alignments to Improve de Novo Gene Finding.” Bioinformatics 24 (5). Oxford University Press: 637--44.↑

[4] Stanke, Mario, Oliver Schöffmann, Burkhard Morgenstern, and Stephan Waack. 2006. “Gene Prediction in Eukaryotes with a Generalized Hidden Markov Model That Uses Hints from External Sources.” BMC Bioinformatics 7 (1). BioMed Central: 62.↑