Pipeline designed for Vibrio genus

Installation

Dowload the pipeline using the command: git clone https://github.com/EnvGen/Degprimer_design

The pipeline uses the programs:

snakemake 3.13.3

muscle v3.8.1551

Degeprime v1.1.0

mfeprimer 3.2.3

Create conda environment

conda create -n primers -c bioconda snakemake=3.13.3 muscle=3.8.1551

Degeprime and MFEprimer must be manually downloaded if you want to use a different version.

Usage

set pipeline parameters in config_primer_design.json using the command: nano config_primer_design.json and modify the parameters and save changes by taping ctrl x and tape y:

  "work_dir": "/absolute/path/to/primer_design/",

  "tar_gff_files": "/absolute/pat/to/genome_gff_files.tar",   -- tar file downloaded from NCBI, "no required if "gene_sequences" provided --

  "tar_file_genomes": "/absolute/path/to/genome_fasta.tar",   -- tar file downloaded from NCBI, "no required if "gene_sequences" provided --

  "NCBI_gene_name": "your option", -- NCBI name of the target gene (required), e.g., "GroEL", "'16S ribosomal RNA'"  --

  "NCBI_gene_region": "your option", -- standard NCBI region name of the target gene (required), e.g., "CDS", "exon"  --

  "gff_file": "/absolute/path/to/gff_with_selected_gene_annotation.gff", -- if file doesn't exits, it will be generated using "tar_gff_files" --

  "gene_sequences": "/absolute/path/to/gene_sequeneces.fna",  -- if not provided it will be generated using "tar_gff_files", "tar_file_genomes", and "gff_file" --

  "muscle_params": "-maxiters 3",

  "trim_degeprime": "-min 0.9",

  "degeneracies": "4,12",

  "primer_sizes": "17,18,19,20,21,22",

  "primer_selection_parameters": "-c 0.99 -g 40", -- -c stands for coverage (fraction) and -g for minimum GC content (%) --

  "amplicon_size": "50,550" -- minimum and maximum amplicon size --

  "threads": 6, -- Number of CPUs to be used --
  
  	  "Output_suffix_name": "your option", -- Remember to rename it if you changed one of the parameters --

run the pipeline using the commands:

`conda activate primers`
`snakemake -s design_primer --cores <number of threads>`

output

Directory Results:

	file "List_<Output_suffix_name>": Lists all possible primer pairs combinations of selected primers

	file "primers_<Output_suffix_name>_table": Lists of selected primers. It includes primer name, sequences, reverse complement, degeneracy, length, GC range, TM range

intermediare output

Directory MSA:

It contains the multiple sequence alignment generated by muscle file `MSA_<Output_suffix_name>`, and the file `timmed_align_file_from_MSA_<Output_suffix_name>` generated by Degeprime

Directory Potential_primers:

It contains the files generated by MFEprimer (dimer and hairpin primer evaluation) and by Degeprime