# toxolib

A Python package for metagenomic taxonomic profiling and abundance matrix generation.

## Installation

### Using pip

```bash
pip install toxolib
```

### Install directly from GitHub

```bash
pip install git+https://github.com/dhruvac29/toxolib.git
```

### Using conda

We recommend using conda to install all dependencies. An environment file is included in the package:

```bash
# Clone the repository
git clone https://github.com/dhruvac29/toxolib.git
cd toxolib

# Create and activate the conda environment
conda env create -f environment.yml
conda activate taxonomy_env

# Install the package
pip install -e .
```

## Requirements

This package requires the following external tools to be installed and available in your PATH:

- Kraken2
- Bracken
- Krona (for visualization)
- fastp (for preprocessing)
- bowtie2 (for host removal)
- samtools

All these dependencies are included in the conda environment file.

## Database Setup

### Automated Database Setup

Toxolib provides automated database setup for both local and HPC environments.

#### Local Database Setup

```bash
# Set up both Kraken2 and corn genome databases
toxolib db-setup -o /path/to/databases --kraken --corn

# Set up only Kraken2 database
toxolib db-setup -o /path/to/databases --kraken

# Set up only corn genome database
toxolib db-setup -o /path/to/databases --corn

# Force re-download of databases even if they exist
toxolib db-setup -o /path/to/databases --kraken --corn --force
```

After setup, you should set the environment variable for Kraken2:

```bash
export KRAKEN2_DB_DIR=/path/to/databases/Kraken2_DB
```

#### HPC Database Setup

When submitting jobs to the HPC, you can automatically download and set up the databases locally and upload them to the HPC:

```bash
# Automatically download locally and upload both databases to the HPC
toxolib hpc -r sample1_L001_R1.fastq.gz sample1_L001_R2.fastq.gz sample1_L002_R1.fastq.gz sample1_L002_R2.fastq.gz -o /path/on/hpc/output_dir \
    --setup-kraken-db --setup-corn-db

# Automatically download locally and upload only Kraken2 database
toxolib hpc -r sample1_L001_R1.fastq.gz sample1_L001_R2.fastq.gz sample1_L002_R1.fastq.gz sample1_L002_R2.fastq.gz -o /path/on/hpc/output_dir \
    --setup-kraken-db

# Automatically download locally and upload only corn genome database
toxolib hpc -r sample1_L001_R1.fastq.gz sample1_L001_R2.fastq.gz sample1_L002_R1.fastq.gz sample1_L002_R2.fastq.gz -o /path/on/hpc/output_dir \
    --setup-corn-db
```

When using these options, toxolib will:
1. Download the databases to your local machine
2. Extract the databases locally
3. Upload the extracted databases to the HPC
4. Configure the Snakefile to use the correct database paths

This approach works even if your HPC has restricted internet access or firewalls that prevent direct downloads.

### Manual Database Setup

If you prefer to set up the databases manually, you can follow these steps:

#### Kraken2 Database

You can download the standard Kraken2 database from:
[https://genome-idx.s3.amazonaws.com/kraken/k2_standard_20240112.tar.gz](https://genome-idx.s3.amazonaws.com/kraken/k2_standard_20240112.tar.gz)

```bash
wget https://genome-idx.s3.amazonaws.com/kraken/k2_standard_20240112.tar.gz
tar -xzf k2_standard_20240112.tar.gz -C /path/to/kraken2/database
export KRAKEN2_DB_DIR=/path/to/kraken2/database
```

#### Corn Genome Database

For host removal, you can download the corn genome reference from:
[https://glwasoilmetagenome.s3.us-east-1.amazonaws.com/corn_db.zip](https://glwasoilmetagenome.s3.us-east-1.amazonaws.com/corn_db.zip)

```bash
wget https://glwasoilmetagenome.s3.us-east-1.amazonaws.com/corn_db.zip
unzip corn_db.zip -d /path/to/corn_db
```

## Usage

### Local Usage

#### Generate abundance matrix from raw data

```bash
toxolib abundance -r raw_data_1.fastq.gz raw_data_2.fastq.gz -o output_directory
```

This will:
1. Run Kraken2 on the raw data
2. Run Bracken on the Kraken2 results
3. Generate an abundance matrix from the Bracken results

#### Create abundance matrix from existing Bracken files

```bash
toxolib matrix -i sample1_species.bracken sample2_species.bracken -o abundance_matrix.csv
```

### HPC Usage

Toxolib can run the analysis pipeline on an HPC cluster using SLURM for job scheduling.

#### 1. Set up HPC connection

```bash
toxolib hpc-setup --hostname your-hpc-server.edu --username your-username --key-file ~/.ssh/id_rsa
```

This will save your HPC connection details to `~/.toxolib/hpc_config.yaml`.

#### 2. Run the pipeline on HPC

```bash
toxolib hpc -r raw_data_1.fastq.gz raw_data_2.fastq.gz -o /path/on/hpc/output_dir \
    --kraken-db /path/on/hpc/kraken2_db \
    --corn-db /path/on/hpc/corn_db \
    --partition normal --threads 32 --memory 200 --time 144:00:00
```

This will:
1. Upload your raw data files to the HPC
2. Create a Snakemake workflow file
3. Upload an environment.yml file to the HPC
4. Submit a SLURM job to run the analysis
5. Return a job ID for tracking

##### Automatic Conda Environment Creation

When submitting a job to the HPC, toxolib will automatically:
1. Upload a conda environment.yml file to the HPC
2. Create a conda environment in the output directory if it doesn't exist
3. Activate the environment before running the analysis

This ensures all required dependencies are available on the HPC without requiring manual environment setup.

#### 3. Check job status

```bash
toxolib hpc-status --job-id your_job_id
```

#### 4. Download results when complete

```bash
toxolib hpc-download --job-id your_job_id --output-dir ./local_results
```



#### Manual Setup on HPC

When using the HPC functionality, you can manually upload and extract these databases on your HPC system:

```bash
# On your local machine, download the databases
wget https://genome-idx.s3.amazonaws.com/kraken/k2_standard_20240112.tar.gz
wget https://glwasoilmetagenome.s3.us-east-1.amazonaws.com/corn_db.zip

# Upload to HPC (using scp)
scp k2_standard_20240112.tar.gz your-username@your-hpc-server.edu:/path/on/hpc/
scp corn_db.zip your-username@your-hpc-server.edu:/path/on/hpc/

# SSH into HPC and extract
ssh your-username@your-hpc-server.edu
mkdir -p /path/on/hpc/kraken2_db
tar -xzf /path/on/hpc/k2_standard_20240112.tar.gz -C /path/on/hpc/kraken2_db
mkdir -p /path/on/hpc/corn_db
unzip /path/on/hpc/corn_db.zip -d /path/on/hpc/corn_db
```

Then when running toxolib, specify these paths:

```bash
toxolib hpc -r raw_data_1.fastq.gz raw_data_2.fastq.gz -o /path/on/hpc/output_dir \
    --kraken-db /path/on/hpc/kraken2_db \
    --corn-db /path/on/hpc/corn_db
```

## License

MIT
