ToxCodAn-Genome is a computational tool designed to annotate toxin genes in genomes of venomous lineages.
The guide for toxin annotation in genomes is here.
Clone the ToxCodAn-Genome respository and add the bin folder into your PATH:
git clone https://github.com/pedronachtigall/ToxCodAn-Genome.git
export PATH=$PATH:$PWD/ToxCodAn-Genome/bin/
echo "export PATH=$PATH:$PWD/ToxCodAn-Genome/bin/" >> ~/.bashrcto add it permanently.- Replace
~/.bashrcto~/.bash_profileif needed.
- Replace
- Apply "execution permission" to executables if needed:
chmod +x $PWD/ToxCodAn-Genome/bin/*
- Python, biopython, and pandas
- NCBI-BLAST (v2.9.0 or above)
- Exonerate
- Miniprot
- GffRead
- Hisat2 - Optional (used in Transcriptome assembly)
- Samtools - Optional (used in Transcriptome assembly)
- StringTie - Optional (used in Transcriptome assembly)
- Trinity - Optional (used in Transcriptome assembly)
- SPAdes - Optional (used in Transcriptome assembly)
Ensure that all requirements are working properly.
- Tip: First, ensure that you have added all conda channels properly in the following order:
conda config --add channels defaultsconda config --add channels biocondaconda config --add channels conda-forge
- Create the environment:
conda create -n ToxcodanGenome -c bioconda python biopython pandas blast exonerate miniprot gffread hisat2 samtools stringtie trinity spades
- Git clone the ToxCodAn-Genome repository and add the bin to your PATH:
git clone https://github.com/pedronachtigall/ToxCodAn-Genome.gitecho "export PATH=$PATH:$PWD/ToxCodAn-Genome/bin/" >> ~/.bashrc- Replace
~/.bashrcto~/.bash_profileif needed.
- Replace
- It may be needed to apply "execution permission" to all bin executables in "ToxCodAn-Genome/bin/":
chmod +x ToxCodAn-Genome/bin/*
- Then, run ToxCodAn-Genome as described in the "Usage" section.
- To activate the environment to run ToxCodAn-Genome just use the command:
conda activate ToxcodanGenome - To deactivate the environment just use the command:
conda deactivate
We have designed toxin databases for some venomous lineages that were tested and working well.
The toxin databases with a brief descriptions are available in the "Databases" folder.
You can check the guide to learn how to design a custom toxin database (when venom tissue transcriptomic data is available or not).
The toxin database must be set with the -d option (see below).
Usage: toxcodan-genome.py [options]
Options:
-h, --help show this help message and exit
-g fasta, --genome=fasta
Mandatory - genome sequence in FASTA format,
/path/to/genome.fasta
-d fasta, --database=fasta
Mandatory - database with coding sequences (CDSs) of
toxins in FASTA format, /path/to/cds.fasta
-C fasta, --cds=fasta
Optional - toxin coding sequences (CDSs) of the
individual/species previously annotated from de-
novo/genome-guided assembly in FASTA format,
/path/to/toxin_cds.fasta
-t fasta, --transcripts=fasta
Optional - transcripts recovered from venom tissue
RNA-seq data for the individual/species using de-
novo/genome-guided assembly methods in FASTA format,
/path/to/transcripts.fasta
-r fastq(.gz), --reads=fastq(.gz)
Optional - pre-processed reads (i.e., adapters trimmed
and low-quality reads removed) obtained from the toxin
tissue of the species in FASTQ(.GZ) format. If single-
end (or merged reads), specify only one file (e.g.,
path/to/reads.fastq). If paired-end, specify both
files in a comma-separated format (e.g.,
path/to/reads_1.fastq,path/to/reads_2.fastq). If you
also set transcripts file in the "-t" parameter, this
parameter/file will be ignored.
-u fasta, --uniprot=fasta
Optional - path to uniprot/toxprot database to perform
an extra step of similarity search to compare the
annotated toxins to the uniprot/toxprot database. It
will output a report file in TXT format containing the
annotated toxin and the best hit in the
uniprot/toxprot database, alongside their percent
identity, alignment length, toxin length, and
uniprot/toxprot entry length.
-o folder, --output=folder
Optional - output folder, /path/to/output_folder; if
not defined, the output folder will be set in the
current directory with the following name
[ToxCodAnGenome_output]
-p int, --percid=int Optional - threshold value used as the minimum percent
identity between match CDSs and genome [default=80]
-s int, --mingenesize=int
Optional - threshold value used as the minimum size of
a gene [default=400]
-S int, --maxgenesize=int
Optional - threshold value used as the maximum size of
a gene [default=50000]
-l int, --length=int Optional - minimum size of a CDS; it will remove any
annotated CDS shorter than the specified threshold
[default=200]
-k boolean value, --keeptemp=boolean value
Optional - keep temporary files. Use True to keep all
temporary files or False to remove them
[default=False]
-c int, --cpu=int Optional - number of threads to be used in each step
[default=1]
Basic usage:
toxcodan-genome.py -g genome.fasta -d toxin_database.fasta
Check our tutorial to learn how to use ToxCodAn-Genome.
Check our guide to have full details about running ToxCodAn-Genome and how to perform toxin annotation in genomes.
ToxCodAn-Genome has the following inputs as mandatory:
- The genome in FASTA format through the
-goption. - The toxin database in FASTA format through the
-doption.
By default, ToxCodAn-Genome outputs the following files:
ToxCodAnGenome_output/
├── annotation_removed.txt
├── annotation_warning.txt
├── matched_GTFs/
│ ├── contig_1--1234-5678.gtf
│ ├── contig_2--11234-15678.gtf
│ ├── ...
│ └── contig_N--111234-115678.gtf
├── matched_regions.gtf
├── toxin_annotation_cds.fasta
├── toxin_annotation.gtf
└── toxin_annotation_pep.fasta
Description of the output files:
toxin_annotation -> final toxin annotation files (including a gtf and two fasta files of CDSs and peptides)
annotation_warning.txt -> list of annotations in the final toxin annotation file that need manual inspection (may represent truncated paralogs, pseudogenes, and/or erroneous annotations)
annotation_removed.txt -> annotations that were removed from the final toxin annotation file (may represent erroneous/incomplete annotations)
matched_regions.gtf -> regions of genome matching to full-length toxin CDSs in the database (that returned or not a toxin annotation)
matched_GTFs/ -> folder containing all matched regions that returned a toxin annotation (each file is named by the genome position as follows "contig--start-end")
If you want to keep all temporary files, run ToxCodAn-Genome with the parameter -k True.
If you use or discuss ToxCodAn-Genome, its guide, or any script available at this repository, please cite:
Nachtigall et al. (2024) ToxCodAn-Genome: an automated pipeline for toxin-gene annotation in genome assembly of venomous lineages. Giga Science. DOI: https://doi.org/10.1093/gigascience/giad116
🐛🆘💬
To report bugs, to ask for help and to give any feedback, please contact Pedro G. Nachtigall: [email protected]
[Q1] What Operation System (OS) do I need to use ToxCodAn-Genome?
- We tested ToxCodAn-Genome in Linux Ubuntu 16, 18 and 20. However, we believe that ToxCodAn-Genome should work on any UNIX OS able to have all dependencies installed.
[Q2] How long will take to ToxCodAn-Genome finish the analysis?
- We tested ToxCodAn-Genome using a personal computer (6-Core i7 with 16Gb memory) and 6 threads (
-c 6). It took less than 2 minutes to finish the analysis using a genome with an average size of 1.6Gb. If the user has more threads available for use, the running time will decrease.
[Q3] ToxCodAn-Genome is returning an error in the "generating final output" step similar to subprocess.CalledProcessError and Segmentation fault (core dumped). What should I do?
- This error can be caused by one or more lines containing a huge sequence. Some tools and packages, like Bio::DB::Fasta used by GffRead, can't process a fasta file with lines containing more than 65,536 characters. So, if you have any large sequence in one unique line, do the following:
- download the script BreakLines.py to "break" the genomic sequences into 100 nts per line
wget https://raw.githubusercontent.com/pedronachtigall/CodAn/master/scripts/BreakLines.py- run BreakLines script:
python BreakLines.py genome.fasta genome_breaklines.fasta - use the "genome_breaklines.fasta" to run ToxCodAn-Genome. It will solve this issue.