[](https://edwards.flinders.edu.au)
[](https://zenodo.org/doi/10.5281/zenodo.8365088)
[](https://opensource.org/licenses/MIT)
[](https://github.com/beardymcjohnface/Snaketool)
[](https://github.com/linsalrob/spae/actions/workflows/testing.yml)
# Sphae
## Phage toolkit to detect phage candidates for phage therapy
<p align="center">
<img src="sphae.png#gh-light-mode-only" width="300">
<img src="sphaedark.png#gh-dark-mode-only" width="300">
</p>
**Overview**
This snakemake workflow was built using Snaketool [https://doi.org/10.1371/journal.pcbi.1010705], to assemble and annotate phage sequences. Currently, this tool is being developed for phage genomes. The steps include,
- Quality control that removes adaptor sequences, low-quality reads and host contamination (optional).
- Assembly
- Contig quality checks; read coverage, viral or not, completeness, and assembly graph components.
- Phage genome annotation'
- Annotation of the phage genome
A complete list of programs used for each step is mentioned in the sphae.CITATION file.
### Install
**Pre-requisites**
- gcc
- conda
- libgl1-mesa-dev (ubuntu- for Bandage)
- libxcb-xinerama0 (ubuntu- for Bandage)
**Install**
Setting up a new conda environment
```bash
conda create -n sphae python=3.11
conda activate sphae
conda install -n base -c conda-forge mamba #if you don't already have mamba installed
```
Steps for installing sphae workflow
```bash
#clone sphae repository
git clone https://github.com/linsalrob/sphae.git
#move to sphae folder
cd sphae
#install sphae
pip install -e .
#confirm the workflow is installed by running the below command
sphae --help
```
## Installing databases
Run command,
```
sphae install
```
Install the databases to a directory, `sphae/workflow/databases`
This workflow requires the
- Pfam35.0 database to run viral_verify for contig classification.
- CheckV database to test for phage completeness
- Pharokka databases
- Phynteny models
This step takes approximately 1hr 30min to install and requires 9G of storage
## Running the workflow
The command `sphae run` will run QC, assembly and annotation
**Commands to run**
Only one command needs to be submitted to run all the above steps: QC, assembly and assembly stats
```bash
#For illumina reads, place the reads both forward and reverse reads to one directory
sphae run --input tests/data/illumina-subset --output example -k
#For nanopore reads, place the reads, one file per sample in a directory
sphae run --input tests/data/nanopore-subset --sequencing longread --output example -k
#To run either of the commands on the cluster, add --profile slurm to the command. For instance here is the command for longreads/nanopore reads
#Before running this below command, make sure have slurm config files setup, here is a tutorial, https://fame.flinders.edu.au/blog/2021/08/02/snakemake-profiles-updated
sphae run --input tests/data/nanopore-subset --preprocess longread --output example --profile slurm -k
```
**Output**
Output is saved to `example/RESULTS` directory. In this directory, there will be four files
- Genome annotations in GenBank format (Phynteny output)
- Genome in fasta format (either the reoriented to terminase output from Pharokka, or assembled viral contigs)
- Circular visualization in `png` format (Pharokka output)
- Genome summary file
Genome summary file includes the following information to help,
- Sample name
- Length of the genome
- Coding density
- If the assembled contig is circular or not (From the assembly graph)
- Completeness (calculated from CheckV)
- Contamination (calculated from CheckV)
- Taxonomy accession ID (Pharokka output, searches the genome against INPHARED database using mash)
- Taxa mash includes the number of matching hashes of the assembled genome to the accession ID/Taxa name. Higher the matching hash- more likely the genome is related to the taxa predicted
- Gene searches:
- Whether integrase is found (search for integrase gene in annotations)
- Whether anti-microbial genes were found (Pharokka search against AMR database)
- Whether any virulence factors were found (Pharokka search against virulence gene database)
- Whether any CRISPR spacers were found (Pharokka search against MinCED database)
## Issues and Questions
This is still a work in progress, so if you come across any issues or errors, report them under Issues.
Raw data
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