ClosTyper

⚠️Please note that clostyper is still in an early stage of development. There may be substantial changes as many features are still being added

ClosTyper is a pipeline tool designed to automate characterization and genotyping of selected Clostrdia species using the whole genome sequencing data
ClosTyper is written in Snakemake that allows reproducibility and scalability of the intergrated workflow.

✋ This tool is under active development ✋
You may think of use WGSBAC (https://gitlab.com/FLI_Bioinfo/WGSBAC) that combine different generalized modules for bacterial characterization based on WGS data

Requirements

Software requirements

before start, you need to make sure that the follwoing software are installed in your system and that they are available in your $PATH

1. any2fasta: Convert various sequence formats to FASTA (https://github.com/tseemann/any2fasta)

   • Installation instructions: https://github.com/tseemann/any2fasta#github
   • Required version: 0.4.2 ro later

2. snakemake: a workflow management system that creates reproducible and scalable data analyses. Snakemake workflows can entail a description of required software, which will be automatically deployed to any execution environment (https://snakemake.github.io/)

   • Installation instructions: https://snakemake.readthedocs.io/en/stable/getting_started/installation.html
   • Required version: 6.15.5 or later

3. pigz: A parallel implementation of gzip for modern multi-processor, multi-core machines (https://zlib.net/pigz/)

   • Installation instructions: https://zlib.net/pigz/
   • Required version: 2.3.4 or later

4. conda: package, dependency and environment management system (https://docs.conda.io/en/latest/)

   • Installation instructions: https://docs.conda.io/projects/conda/en/latest/user-guide/install/linux.html

Databases requirements

1. (Mini)Kraken2 database: taxonomic profiling of sequencing data (https://ccb.jhu.edu/software/kraken2/)
   • Download page: https://ccb.jhu.edu/software/kraken2/downloads.shtml

Installation

Install clostyper from source

These instructions will install the latest version of ClosTyper:

git clone https://gitlab.com/FLI_Bioinfo/clostyper.git  
ln -s `pwd`/clostyper/bin/clostyper /usr/local/bin/ # choose a folder in your $PATH

This should be the directory structure of clostyper

|-- bin
|   `-- clostyper
|-- config
|   `-- README.md
|-- dbs
|   |-- custom_dbs
|   `-- trimmomatic.fa
|-- README.md
`-- workflow
    |-- envs
    |-- rules
    |-- scripts
    `-- Snakefile

Usage

First execution

On first execution of clostyper with clostyper -h, a config file will be automatically created <clostyper_config.txt> under the folder <clostyper/config>.
This file should include the paths to databases and schemes to be used with clostyper.
✍ Important: at least the full path to the Kraken2 database must be provided.

Basic usage [with test data]

download test data:

wget -O test_data_clostyper.tar.gz https://zenodo.org/record/6656045/files/test_data_clostyper.tar.gz?download=1 
tar xzvf test_data_clostyper.tar.gz 

Run clostyper

✍ Note: the full path to the Kraken2 database should have been specified in the config file <clostyper/config/clostyper_config.txt>. If not, you must use --kraken2db </path/2/kraken_database/>

• To check raw data quality
  This will only execute the fastp & kraken2
  Basic call: clostyper --check_quality -d [FASTQ_DIRECTORY] -r [REFERENCE] [-o WORKING_DIRECTORY]

• With the test data

clostyper --check_quality -d test_data/input_data/ -r test_data/input_data/ref.gbk -o results_dir -A -T <cpus> 

• To configure and execute the full pipeline
  ✍ WARNING: Many features of clostyper are still experimental. Please report issues in the issue tracker (https://gitlab.com/FLI_Bioinfo/clostyper/-/issues)
  This will execute the full pipeline
  Basic call: clostyper -d FASTQ/FASTA_DIRECTORY -r REFERENCE [-o WORKING_DIRECTORY] [-s SPECIES]

• With the test data

clostyper -d test_data/input_data/ -r test_data/input_data/ref.gbk -o results_dir -A -T <cpus> 

• To search C. difficile for mobile elements
  To ONLY search your genomes for Clostridium difficile mobile elements, invoke the flag --run_only_species_wf together with flag -s

• Example with the test dataset

clostyper -d test_data/input_data/ -r test_data/input_data/ref.gbk -o results_dir -s cdifficile --run_only_species_wf -A -T <cpus>

Full usage options

> clostyper -h
ClosTyper: Clostridia characterization and typing pipeline

Version: 0.1-beta (available at: https://gitlab.com/FLI_Bioinfo/ClosTyper)
USAGE:
 ---------------------
     clostyper --check_quality -d FASTQ_DIRECTORY [-o WORKING_DIRECTORY]
     clostyper -d FASTQ/FASTA_DIRECTORY -r REFERENCE [-o WORKING_DIRECTORY] [-s SPECIES] [--run_cgmlst]
     clostyper -t SAMPLE_TABLE -r REFERENCE [-o WORKING_DIRECTORY] [-s SPECIES] [--run_cgmlst]
 ---------------------
INPUT:
   -d, --fastx-directory          DIR, a directory where fastq reads or assembled genomes are present. Required unless -t flag was used
                                    Format: [ID]_{1,2}.fastq{.gz} [ID]_S*_R{1,2}_001.fastq{.gz} [ID]_R{1,2}.fastq{.gz} OR [ID].{fasta,fna,fa}
   -t, --sample_table             FILE (tab delimited), a four-columns based table. See an example in the documentation!
                                    Required unless the -d flag was used. If -t and -d flags were activated, -d will be ignored
   -r, --reference                FILE, Reference genome. Format: {ID}.{gbk,fasta,gff,embl} (required)
   -s, --species                  Run species-specific workflow (default: False; run only the general workflow)
                                    Currently supported Clostridia species are: cdifficile
OUTPUT:
   -o, --output-directory         DIR, output directory for the snakemake results (default: output_dir_[timestamp]/)
   -w, --overwrite                Overwrite an existing directory with the results. Useful to append results to previous runs
   -q, --quiet                    Suppress clostyper messages. Report only warnings, errors and the snakemake call
WORKFLOW:
   -Q, --check_quality            Quickly perform quality assurance on Illumina data [recommended before doing analysis] [EXPERIMENTAL]
   --run_cgmlst                   Do cgMLST analyis using the chewiesnake pipeline [EXPERIMENTAL]
   --run_only_species_wf          Execute ONLY the specified species-specific workflow. Require '-s' [EXPERIMENTAL]
   --select_reference             Select appropriate reference for the SNP anlysis of the dataset [EXPERIMENTAL]
   --snp_pipeline                 Select which pipeline to call SNPs [EXPERIMENTAL]
                                    Supported SNP pipelines are: snippy, reddog, nasp, cfsanpipeline
   --kraken2db                    Path to (Mini)kraken2 DB
   --disable_pangenome            Disable the pangenome analyis [EXPERIMENTAL]
   --disable_report               Do not make the html report [EXPERIMENTAL]
OTHERS:
   -A, --autorun                  Automatically run snakemake workflow after configuration (default: False)
   -T, --threads                  Number of threads to use (default: 16)
   --check_dep                    Check if dependencies are ok and then exit
   --no-color                     Do not use a colored output (default: False)
HELP:
   -h, --help                     Show this help and exit
   --help_all                     Show extended help for all software settings options [EXPERIMENTAL]
   --version                      Show clostyper's version number and exit
   --citation                     Show clostyper's citation and exit