jkimlab/plrgen repository overview

⁠Overview

PLR-GEN is a tool for the generation of pseudo-long-reads (PLRs) by using short-reads of a metagenomic sample and microbial reference genome sequences as input.

You can see PLR-GEN Github⁠

⁠USAGE

⁠Input preparation

1. Input single-end or paired-end NGS short reads

   • Read sequence files are mandatory with fastq format (or compressed by .gz file)
   • You can use single-end reads with -s (incompatible with -1 and -2)
   • Or you can use paired-end reads with -1 and -2 (incompatible with -s)

2. A list of reference genome sequences

   • Reference genome sequence files are mendatory with fasta format (or compressed by .gz file)
   • You can use -r or -ref option (incompatible with -tama)with a list of all the file paths of reference genomes to set up the references for running PLR-GEN

3. (Additional) If TAMA is installed, you can predict putative bacterial species in your dataset using TAMA, running PLR-GEN using -tama option (incompatible with -r).

⁠Running options of PLR-GEN

• Running PLR-GEN

    PLR-GEN.pl [options] -1 <pe1> -2 <pe2> (or -s <se>) -r <ref_list> -o <out_dir>
    
  

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1. Input options

   • -s : an input sequence file with unpaired reads (single-end reads); a fastq or fastq.gz file
   • -1 and -2 : input sequence files with paired-end reads; fastq or fastq.gz files
   • -r or -ref : a list with all file paths of reference genome sequences; a text file
   • -tama : if this option is used, TAMA program will be used for reference preparation instead of -r (default: not used)
   • -sampling : if this option is used, reference genomes will be randomly selected and used (default: not used)

2. Output options

   • -o : a path of output directory (default: ./PR_out; final output: ./PR_out/pseudo-long-read.fa)
   • -t : if this option is used, all intermediated output files are saved (default: not used)

3. Running options

   • -p : the number of threads
   • -l or -min_length : minimum length cutoff of generated pseudo-long reads (PLRs) (default: 100)
   • -q or -mapq : cutoff of mapping quality (used in samtools) (default: 20)
   • -c or -min_count : cutoff of mapping depth for generating normal nodes and bubbles; after piling-up of read mapping, alignment column less than the cutoff value will be discarded (default: 1)
   • -d or -min_depth : cutoff of mapping depth of bubbles for filtering bubble nodes; a bubble with less than the cutoff % of mapping depth will be converted to a normal node

• Help page of PLR-GEN

    Usage: PLR-GEN.pl [options] -1 <pe1> -2 <pe2> (or -s <se>) -r <ref_list> -o <out_dir>
  
    == MANDATORY 
    -s	<se>	File with unpaired reads [incompatible with -1 and -2]
    -1	<pe1>	File with #1 mates (paired 1) [incompatible with -s]
    -2	<pe2>	File with #2 mates (paired 2) [incompatible with -s]
    -r|-ref	<ref_list>	The list of reference genome sequence files
    -tama	Reference preparation using TAMA [incompatible with -r|-ref]
    -sampling	<proportion>	proportion to random sampling for references (default: off, range: 0-1)
    -o	<out_dir>	Output directory (default: ./PR.out)
  
    ==Running and filtering options
    -p|-core	<integer>	The number of threads (default: 1)
    -q|-mapq	<integer>	Minimum mapping quality (default: 20)
    -l|-min_length	<integer>	Cutoff of minimum length of pseudo-long reads (default: 100bp)
    -c|-min_count	<integer>	Cutoff of minimum mapping depth for each node (default: 1)
    -d|-min_depth	<integer> Cutoff of mapping depth of bubbles (default: 1, range: 0-100)
    		0: all bubbles are used.
    		1: bubbles with less than 1% mapping depth from mapping depth distribution of bubbles are converted to normal nodes.
    		100: all bubbles are converted to normal nodes
  
    ==Other options
    -t|-temp	If you use -t option, all intermediate files are left.
    	Please careful to use this option because it has to be needed very large space.
    -h|-help	Print help page.
  

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⁠(Additional) TAMA installation

• For running PLR-GEN, if you want to use TAMA with -tama option for preparation of reference genomes, you can install TAMA using below commands. It will automatically download and install TAMA into the given path, and set ready-made species-level TAMA databases. These TAMA databases require total 300GB disk space, so please carefully set up the path for installation of TAMA. If you do not specify a path for TAMA, the TAMA package and TAMA databases will be set inside "bin" directory in the PLR-GEN.

1. Installation of TAMA with Docker

    docker run --rm -v /PATH/TO/TAMA_DIR:/tama_dir -t jkimlab/plrgen:latest /work_dir/src/TAMA_install.pl /tama_dir
    
   

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2. Manual installation

• You can use manually installed TAMA. To use -tama option for PLR-GEN, you need to install TAMA and ready-made species-level databases into /LOCAL_DISK/PLR-GEN/bin/ directory or make a link of path of installed TAMA directory to /LOCAL_DISK/PLR-GEN/bin/ directory.

For more information of TAMA, see TAMA Github⁠

⁠Examples of running PLR-GEN

• Suppose (1) you pulled the PLR-GEN docker image (jkimlab/plrgen), (2) read_1.fq, read_2.fq are in /LOCAL_DISK/DATA directory, and (3) all reference sequence files are in /LOCAL_DISK/DATA/fasta/ directory. In this situation, you can run PLR-GEN with docker image as followed command,

  1. Make a reference list file in the /LOCAL_DISK/DATA directory with the file system for the docker image, for an example,

     file: /DATA/reference_list.txt

      /data_dir/fasta/REF_1.fa
      /data_dir/fasta/REF_2.fa
      /data_dir/fasta/REF_3.fa
     

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  2. Run the docker image, mounting /DATA directory to /data_dir directory of docker image, for an example,

      docker run -v /LOCAL_DISK/DATA:/data_dir -t jkimlab/plrgen PLR-GEN.pl -1 /data_dir/read_1.fq -2 /data_dir/read_2.fq -r /data_dir/reference_list.txt -o /data_dir/output
      
     

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• If you want to use TAMA installed at /LOCAL_DISK/PROGRAMS/TAMA, you need to mount the TAMA directory to /work_dir/bin/ directory of docker image, for an example,

    docker run -v /LOCAL_DISK/PROGRAMS/TAMA:/work_dir/bin/TAMA -v /LOCAL_DISK/DATA:/data_dir -t jkimlab/plrgen PLR-GEN.pl -1 /data_dir/read_1.fq -2 /data_dir/read_2.fq -r /data_dir/reference_list.txt -o /data_dir/output
  

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