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BEAV: Bacterial Element Annotation reVamped

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# beav - a bacterial genome and mobile element annotation pipeline

<img src="https://github.com/weisberglab/beav/blob/main/beav_logo.jpg" width=25% height=25%> 
beav: Bacteria/Element Annotation reVamped

**beav** is a command line tool that streamlines bacterial genome and mobile genetic element annotation. It combines multiple annotation tools, automating the process of running, parsing, and combining the results into a single easy-to-read output. Annotated features include secretion systems, anti-phage defense systems, integrative & conjugative/mobilizable elements, integrons, prophage regions, amino acid biosynthesis pathways, small carbon metabolite catabolism pathways, and biosynthetic gene clusters. Type VI secretion system (T6SS) vgrG operons are automatically identified. Plasmid origin of transfer (oriT) elements are also characterized.

The **beav** pipeline also includes several tools and databases that enhance the annotation of plant associated microbes, including phytopathogens and symbionts. Custom bakta databases provide correct gene names and annotations for phytopathogen virulence genes, effectors, and genes important for mutualist symbiosis. Other tools annotate promoter elements such as the pip box, tts box, nod box, tra box, vir box, etc.

An optional Agrobacterium-specific pipeline identifies the presence of Ti and Ri plasmids and classifies them under the Weisberg et al. 2020 scheme. It also annotates Ti/Ri plasmid elements including T-DNA borders, overdrive, virbox, trabox, and other binding sites, and determines the biovar and genomospecies of the input strain. Virulence and T-DNA genes, including opine synthase and transport/catabolism loci, are also correctly named and annotated.

**beav** will generate Circos plot annotating important features for the genome as well as pTi/pRi plasmid (if Agrobacterium specific analysis is conducted). It is also possible to separately run the Circos script.

<figure>
<img src="https://github.com/weisberglab/beav/blob/main/C58_Genome.png" alt="C58 Genome Circos">
<figcaption>Example Circos plot of whole genome annotations automatically generated by beav. </figcaption>
</figure>
 
<figure>
<img src="https://github.com/weisberglab/beav/blob/main/C58_pTi.png" alt="C58 pTi Plasmid Circos">
<figcaption>Example Circos plot visualizing oncogenic Ti/Ri plasmids generated by the optional Agrobacterium-specific pipeline. </figcaption>
</figure>

# **Quick Start**
```
#download and install beav with conda/mamba
mamba create -n beav beav
conda activate beav
#download all prerequisite databases
beav_db
#run beav
beav --input /path/to/file/test.fna --threads 8 --skip_tiger
```
    
# **Installation**
The **beav** pipeline requires a number of programs and databases be installed. Therefore, it is highly encouraged and recommended to use conda to install **beav** and all of its dependencies.

Once the tool is installed, run the **beav_db** tool to download all necessary databases.

# From conda (Recommended) 
It is recommended to use either conda with [libmamba](https://www.anaconda.com/blog/a-faster-conda-for-a-growing-community) or mamba to install beav as this will greatly speed up the time solving the environment.

instructions for conda:
```
conda create -n beav
conda install -n beav beav
```
alternative instructions using mamba:
```
conda create -n beav
mamba install -n beav beav
```
or as one combined command:
```
conda create -n beav beav
or
mamba create -n beav beav
```

The conda environment can then be activated using:
```
conda activate beav
```

# Alternative: From pixi

Pixi is a new tool for installing and managing conda packages and simplifies many things. To install beav globally, and with no need to activate environments, run the following command:
```
pixi global install beav
```
Then you can then follow the database installation step without running conda activate.

# Alternative: From source

Clone the beav github repository.
```
git clone https://github.com/weisberglab/beav.git
```
**If installing from source, DBSCAN-SWA, TIGER2, and GapMind (PaperBLAST) need to be installed in the software folder within the beav folder. Then the BEAV_DIR environment variable needs to be set and pointing to the beav directory.**

**Prerequisites:**
| Program | Install location |
| --- | --- |
| Bakta | PATH |
| IntegronFinder | PATH |
| MacSyFinder | PATH |
| DefenseFinder | PATH |
| TIGER2 | $BEAV_DIR/software |
| GapMind (PaperBlast) | $BEAV_DIR/software |
| DBSCAN-SWA | $BEAV_DIR/software |
| antiSMASH | PATH |
| EMBOSS | PATH |
| HMMER | PATH |

Databases for each of these programs can then be installed manually. Alternatively, the following can be used to install them automatically.

# Install all databases

```
conda activate beav 
beav_db
```
## Database script optional parameters
    usage: beav_db [--skip_bakta_db] [--light] [--bakta_db_path DIRECTORY] [--update]
        --skip_bakta_db 
            Skip downloading the Bakta databases
        --light
            Install the light version of Bakta databases
        --bakta_db_path DIRECTORY
            Install Bakta databases in nondefault location 
        --update
            Update Bakta databases

# Usage

#### NOTE: If you get an error stating "ModuleNotFoundError: No module named 'nrpys'", then you can run the following command (with the beav conda environment activated) to force reinstall it:
`python -m pip install --upgrade --force-reinstall nrpys`

#### NOTE: there is currently a [bug](https://github.com/mdmparis/defense-finder/issues/42) in the latest DefenseFinder models that cause an error in MacSyFinder when running it. We recommend running Beav with `--skip_defensefinder` until the MacSyFinder [bug fix](https://github.com/gem-pasteur/macsyfinder/commit/27ee21ceb8e7100d9183b084356f791487aca4ad) is released in bioconda. Alternatively, copying the patched file to the MacSyFinder python library folder of your conda release will fix the issue.
<details>
 <summary>Patching instructions</summary>
 To do so, find the python version of your conda environment:

python --version

Then download the patched registries.py file:

wget https://github.com/gem-pasteur/macsyfinder/blob/27ee21ceb8e7100d9183b084356f791487aca4ad/macsypy/registries.py

Then copy it to the correct folder in your conda env, changing the python version as necessary:

cp registries.py $CONDA_PREFIX/lib/python3.9/site-packages/macsypy/
</details>

```
usage: beav [--input INPUT] [--output OUPUT_DIRECTORY] [--strain STRAIN] [--bakta_arguments BAKTA_ARGUMENTS] [--tiger_arguments TIGER_ARGUMENTS][--agrobacterium AGROBACTERIUM] [--skip_macsyfinder] [--skip_integronfinder][--skip_defensefinder] [--skip_tiger] [--skip_gapmind][--skip_dcscan-swa] [--skip_antismash] [--help] [--threads THREADS] [--genbank] [--continue]
    BEAV- Bacterial Element Annotation reVamped
    Input/Output: 
        --input, -i STRAIN.fna
                Input file in fasta nucleotide format (Required)
        --output DIRECTORY
                Output directory (default: current working directory)
        --strain STRAIN
                Strain name (default: input file prefix)
        --bakta_arguments ARGUMENTS
                Additional arguments and database options specific to Bakta 
        --antismash_arguments ARGUMENTS
                Additional arguments and database options specific to antiSMASH (Default: \"$antismash_args\") 
        --tiger_blast_database DBPATH
                Path to a reference genome blast database for TIGER2 ICE analysis (Required unless --skip_tiger is used)
        --run_operon_email EMAIL
                Annotate predicted operons using the Operon-mapper webserver. Must input an email address for the job
    Options:
        --agrobacterium
                Agrobacterium specific tools that identify biovar/species group, Ti/Ri plasmid, T-DNA borders, virboxes and traboxes
        --skip_macsyfinder
                Skip detection and annotation of secretion systems
        --skip_integronfinder
                Skip detection and annotation of integrons 
        --skip_defensefinder
                Skip detection and annotation of anti-phage defense systems 
        --skip_tiger
                Skip detection and annotation of integrative conjugative elements (ICEs)
        --skip_gapmind
                Skip detection of amino acid biosynthesis and carbon metabolism pathways
        --skip_dbscan-swa
                Skip detection and annotation of prophage
        --skip_antismash
                Skip detection and annotation of biosynthetic gene clusters
        --continue
                Continue running BEAV from any point in the pipeline. Rerun programs that gave an error or were skipped.
        --gbk
                Use a GenBank file as input
    General:
        --help, -h
                Show BEAV help message
        --threads, -t
                Number of CPU threads
```
# Options

**--antismash_arguments**

Additional antiSMASH arguments can be input into antiSMASH using the --antismash_arguments option. This allows for full usage of antiSMASH and additional databases.

**--tiger_blast_database**

Required if running TIGER. Users must provide a path to a blast database of reference genomes using the --tiger_blast_database option.

**--bakta_arguments**

Additional arguments can be passed to bakta using the --bakta_arguments option.

**--agrobacterium**

The --agrobacterium option activates an additional pipeline to provide agrobacterium-specific annotation.

**--skip-PROGRAM**

The skip options allow for specified programs to be skipped if the annotation is not needed or required programs are not installed.

**--continue**

The continue option will check the output of existing Beav runs and rerun programs that errored or were skipped. This option allows for the pipeline to be used with existing Bakta runs.

**--gbk**

A GenBank file can be used as the input file when the genbank option is used.

# Examples
**Minimal run**

```
beav --input /path/to/file/test.fna --threads 8 --skip_tiger
```

**Standard run**
```
beav --input /path/to/file/test.fna --threads 8 --tiger_blast_database /path/to/databases/blast/refseq_genomic.fna
```

**Standard run with operon annotation (remote)**
```
beav --input /path/to/file/test.fna --threads 8 --tiger_blast_database /path/to/databases/blast/refseq_genomic.fna --run_operon_email myemail@email.com
```

**Standard run with genbank input**
```
beav --input /path/to/file/test.gbk --threads 8 --tiger_blast_database /path/to/databases/blast/refseq_genomic.fna --gbk
```

**Complex run**

```
beav --input /path/to/file/test.fna --threads 8 --bakta_arguments '--db /path/to/alternative-data-bases/bakta-1.7/' --tiger_blast_database /path/to/databases/blast/allagro.fna --agrobacterium --skip_integronfinder
```

# Standalone Circos plot generation
To generate Circos plots on your GenBank file independant of the **beav** pipeline, make sure the beav conda environment is activated:
```
conda activate beav 
```

Usage:
```
beav_circos -i <GenBank_file> [-c <Contig_for_subset_visualization>] [--pTi <Contig_for_oncogenic_visualization>]
```

Examples:
```
# Generate a general Circos plot for all contigs
beav_circos -i test.gbk

# Generate a general Circos plot for all contigs and a oncogenic Circos plot for single contig
beav_circos -i test.gbk --pTi contig_1

# Generate a general Circos plot for all contigs and a oncogenic Circos plot for a set of contigs
beav_circos -i test.gbk --pTi "contig_1 contig_2"

# Generate a general Circos plot for single contig
beav_circos -i test.gbk -c contig_1

# Generate a general Circos plot for a set of contigs
beav_circos -i test.gbk -c "contig_1 contig_2"
```

# Citation
Beav can be cited as:

Jung, J. M., Rahman, A., Schiffer, A. M., & Weisberg, A. J. (2024). Beav: a bacterial genome and mobile element annotation pipeline. Msphere, 9(8), e00209-24. https://doi.org/10.1128/msphere.00209-24

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