1- Install Docker (https://docs.docker.com/engine/install/ubuntu/)
2- Make Docker run without requiring sudo (https://docs.docker.com/engine/install/linux-postinstall/)
sudo groupadd docker # create the docker group
sudo usermod -aG docker $USER # add your user to the docker group
#Log out and log back in so that your group membership is re-evaluated
docker run hello-world #verify that you can run docker commands without sudo
#If it runs without issues, it means that it works, else please consult the link above for further steps
3- Pull the Braker3 Docker image and test if it runs
docker pull teambraker/braker3
docker run --user 1000:100 --rm -it teambraker/braker3:latest bash
# it should work without sudo if the previous steps worked
4- Install miniconda (https://docs.anaconda.com/miniconda/)
mkdir -p ~/miniconda3
wget https://repo.anaconda.com/miniconda/Miniconda3-latest-Linux-x86_64.sh -O ~/miniconda3/miniconda.sh
bash ~/miniconda3/miniconda.sh -b -u -p ~/miniconda3
rm -rf ~/miniconda3/miniconda.sh
# After installing, initialize your newly-installed Miniconda using the following commands for bash and zsh shells
~/miniconda3/bin/conda init bash
~/miniconda3/bin/conda init zsh
#add conda-forge and bioconda channels to miniconda installation
conda config --add channels bioconda
conda config --add channels conda-forge
conda config --set channel_priority strict
5- Create conda environment called ‘annotation’ and install repeatmodeler
conda create -n annotation bioconda::repeatmodeler
6- Create conda environment called ‘busco_env’ and install Busco
conda create -n busco_env -c conda-forge -c bioconda busco=5.7.1
7- Install interproscan in the base conda environment, make sure the appropriate java and other depndencies are installed:
mkdir ~/my_interproscan
cd ~/my_interproscan
wget https://ftp.ebi.ac.uk/pub/software/unix/iprscan/5/5.69-101.0/interproscan-5.69-101.0-64-bit.tar.gz
wget https://ftp.ebi.ac.uk/pub/software/unix/iprscan/5/5.69-101.0/interproscan-5.69-101.0-64-bit.tar.gz.md5
# recommended checksum to confirm the download was successful: must return *interproscan-5.69-101.0-64-bit.tar.gz: OK*
md5sum -c interproscan-5.69-101.0-64-bit.tar.gz.md5
tar -pxvzf interproscan-5.69-101.0-*-bit.tar.gz
cd ~/my_interproscan/interproscan-5.69-101.0
python3 setup.py -f interproscan.properties # index the hmm models to prepare them into a format used by hmmscan
If you face any of the common issues resolve them as indicated in the interproscan docs. error while loading shared libraries: libgomp.so.1 can be resolved using sudo apt-get install -y libgomp1
8- Install ncbi-genome-download in the base conda environment
pip install ncbi-genome-download
9- Create and environment and install the dependencies for the remove_isoforms module:
mamba create -n remove_isoforms
mamba activate remove_isoforms
pip install biopython
10- Download the appropriate reference protein file from here. Ensure that the reference is unzipped and has appropriate read permissions to be opened with docker: chmod 664 <reference_fasta>
11- Make sure you have read and write permissions to our input and output directories
# reference
bash species_genome_annotation.sh -a <accession_number> -r <reference_fasta> -l <lineage> -o <output_directory> -t <threads>
-a Specify the NCBI accession number for the species genome you want to annotate, must start with GCA_(GeneBank) or GCF_ (RefSeq)
-r Specify the path of the reference fasta file (.fa, .fna, .fasta),
usually obtained from https://bioinf.uni-greifswald.de/bioinf/partitioned_odb11/index.html (for our case Eukaryota.fa.gz)
-l Specify the BUSCO lineage term to be used from this list https://busco.ezlab.org/list_of_lineages.html (for our case euglenozoa)
-o Specify the path of the output directory where the results will be saved, default is working directory
-t Specify the number of threads to use, not recommended above 32, default is 8
-h Display the help message
After species annotation is done, ensure the remove_isoforms directory is present and run:
bash remove_isoforms/remove_isoforms.sh <BASE_NAME>
where BASE_NAME is the name of the directory created by the annotation process.
Use this spreadsheet to track which species must be annotated and use their respective NCBI acession numbers to run the script as indicated below:
# example for Leishmania tarentolae
bash species_genome_annotation.sh -a GCA_033953505.1 -r Eukaryota.fa -l euglenozoa -o ~/tarentolae -t 32