SCAPP assembles plasmids from metagenomic assembly graphs.
SCAPP has been developed and tested on Linux, the installation instructions below have not yet been tested on other operating systems.
This is the preferred installation method
You can install directly from Bioconda with conda install -c bioconda scapp
Alternatively, you can install SCAPP as a conda package (tested with Miniconda3):
Download the installation file install_scapp.yaml in the desired folder. For example:
wget https://raw.githubusercontent.com/Shamir-Lab/SCAPP/master/install_scapp.yaml
Create and activate the conda environment:
conda env create -f install_scapp.yaml
conda activate scapp
Now you can run SCAPP by entering the command scapp.
If not using Conda to install, then download the sources and install according to the following:
SCAPP is written in Python3. SCAPP uses NumPy, NetworkX, pySAM, and nose. The necessary versions of these required dependencies will all be installed by the setup.py script.
SCAPP uses BWA (tested with v0.7.5 and v0.7.17) , NCBI BLAST+ tools (tested with v2.7, v2.9 and v2.10), and samtools (tested with v1.9 and v1.10). The executables of these programs should be available on the system on which SCAPP is run.
The PlasClass classifier should also be installed in order to use the full functionality of SCAPP.
We recommend using a virtual environment. For example, in Linux, before running setup.py:
python -m venv scapp-env
source scapp-env/bin/activate
To install, download and run setup.py:
git clone https://github.com/Shamir-Lab/SCAPP.git
cd SCAPP
python setup.py install
It is possible to install as a user without root permissions:
python setup.py install --user
To install PlasClass, in the folder you would like to install do:
git clone https://github.com/Shamir-Lab/PlasClass.git
cd PlasClass
python setup.py install
Note that this step can be skipped if you installed using Conda.
The BWA, samtools, and BLAST+ executables must be available to SCAPP. They can either be added to your PATH environment variable, or you can specify the paths to each of them in the file scapp/config.json.
For example, if the BWA executable is in /usr/bin/bwa/ then the line "BWA_PATH" : "/usr/bin/bwa", should be completed in the config.json file if that location is not in your PATH.
Once you have completed the above you can run ./run_test.sh from the outermost SCAPP directory to test your installation.
(If the test fails please ensure you have set up the environment, installed, and configured SCAPP as described. Open an issue on this GitHub page with any problems you run into.)
To run the SCAPP pipeline:
scapp -g <fastg graph> -o <output directory> [-k <max k value>] -r1 <reads 1> -r2 <reads 2> [-p <num processes>]
If a BAM alignment file of the reads to the assembly graph already exists, then use the following command to avoid re-running the alignment:
scapp -g <fastg graph> -o <output directory> [-k <max k value>] -b <BAM file> [-p <num processes>]
The common command line options are:
-g/--graph: : Assembly graph fastg file.
-o/--output_dir: Output directory.
-k/max_k: Maximum k value used by the assembler. Default: 55.
-p/--num_processes: Number of processes to use. Default: 16.
-r1/--reads1: Paired-end reads file 1.
-r2/--reads2: Paired-end reads file 2.
-b/--bam: BAM alignment file aligning reads to graph nodes -b and -r1,-r2 are mutually exclusive.
The output files are written in the directory specified by the user:
<prefix>.confident_cycs.fasta is the main output fasta of the plasmid predictions. <prefix> is the name of the assembly graph file (minus the .fastg suffix).
Under the intermediate_files subdirectory <prefix>_cycs.fasta is a fasta file of all cyclic paths that were considered as potential plasmids before filtering to create the subset that is output.
intermediate_files/reads_pe_primary.sort.bam(.bai) is the alignment file for the reads to the assembly graph. It can be re-used with the -b option if SCAPP is re-run on the same sample.
Under the logs subdirectory, the file scapp.log contains all information about the SCAPP run. Other log files in the logs subdirectory may be helpful if there is an error or failure in one of the stages that runs BLAST, BWA, or PlasClass.
More advanced command line options allow for different stages in the SCAPP pipeline to be modified:
-sc/--use_scores: Flag to determine whether to use plasmid scores. Use False to turn off plasmid score use. Default: True.
-gh/--use_gene_hits: Flag to determine whether to use plasmid specific genes. Use False to turn off plasmid gene use. Default: True.
-pc/--plasclass: PlasClass score file. If PlasClass classification of the assembly graph nodes has already been performed, provide the name of the PlasClass output file. (For example: the intermediate_files/plasclass.out file from a previous run of SCAPP).
-pf/--plasflow: PlasFlow score file. To use PlasFlow scores for the nodes instead of PlasClass, provide the name of the PlasFlow output file.-pf,-pc are mutually exclusive.
In addition, all of the different thresholds used in the algorithm can be changed by the user:
-m/--max_CV: Maximum allowed coefficient of variation for coverage. Default: 0.5.
-l/--min_length: Minimum allowed length for potential plasmid. Default: 1000.
-clft/--classification_thresh: Threshold for classifying a potential plasmid as a plasmid. Default: 0.5.
-gm/--gene_match_thresh: Threshold for % identity and fraction of length covered to determine plasmid gene matches. Default: 0.75.
-sls/selfloop_score_thresh: Threshold plasmid score above which a self-loop is considered a potential plasmid. Default: 0.9.
-slm/--selfloop_mate_thresh: Threshold fraction of off-loop mate-pairs, below which a self-loop is considered a potential plasmid. Default:0.1.
-cst/--chromosome_score_thresh: Threshold score, below which a long node is considered a chromosome node. Default: 0.2.
-clt/--chromosome_length_thresh: Threshold length, above which a low scoring node is considered a chromosome node. Default: 10000.
-pst/--plasmid_score_thresh: Threshold score, above which a long node is considered a plasmid node. Default: 0.9.
-plt/--plasmid_length_thresh: Threshold length, above which a high scoring node is considered a plasmid node. Default: 10000.
-cd/--good_cyc_dominated_thresh: Threshold for the maximum fraction of nodes with most mate-pairs off the cycle allowed for the cycleto be considered a potential plasmid. Default: 0.5.
Instead of inputting all of these options on the command-line before each run of SCAPP, the user can change them in the file bin/params.json. Set each variable in this file to the desired value and it will be used in SCAPP. Any value passed as a command-line parameter will override the values set in this file.
SCAPP searches for plasmid-specific genes in the assembly graph and potential plasmids. Curated sets of plasmid-specific genes (see the SCAPP manuscript for details) are located in the scapp/data directory.
The user can add their own plasmid-specific gene sets in this directory. You may put nucleotide gene sequences in the data/nt subdirectory, or amino acid protein sequences in the data/aa subdirectory. The sequence files should be in fasta format.
If you wish to remove certain plasmid-specific sets, simply move them out of the data directory.
SCAPP is designed to run on short, paired-end read libraries (such as Illumina HiSeq) and we believe it will work best in this setting. We have tried to provide support for some requests of running SCAPP with long reads and will add details of how to do this here as we expand support:
The read alignment process used in SCAPP is specific to short paired-end reads. You will have to align your reads to the assembly graph to create a bam file. This alignment file is then provided to SCAPP using the -b option.
MetaFlye generates a *.gfa assembly graph according to a specific definition of the gfa format. This script should convert the gfa file to the fastg format required by SCAPP. The script is run as: python metaflye_gfa2fastg.py . This script is only meant for MetaFlye output (as of Feb 2020).
SCAPP should then be run with this converted fastg file, you bam alignment file AND THE OPTION -k 0.