------------------  Developed by PJ Van Camp  ------------------------

          ||||   ||||||   |||||    |||||   ||   ||  ||||    ||||  
         ||  ||  ||      ||   ||  |||  ||  ||  ||| ||  ||  ||  || 
         |||     ||      ||   ||       ||  ||||||| ||      |||    
           ||    |||||   ||   ||      ||   || | || ||        ||   
            ||   ||      ||   ||    ||     || | || || |||     ||  
             ||  ||      ||   ||   ||      ||   || ||  ||      || 
         |||||   ||||||   |||||   |||||||  ||   ||  ||||   ||||| 
                            ||                                   
                             ||                                  
    ----------    github.com/pieterjanvc/seq2mgs/releases    -------------
   

--- SETUP.SH ---
Run the setup.sh script to verify all dependencies and to test the pipeline.
THe first time a database will be created in the dataAndScripts folder

Arguments [h|t]
 -h Read the help documentation
 -t Run pipeline tests with dummy data (will take bit longer)

The following software needs to be installed:
- SQLite3 
  * Precompiled 32-bit: https://www.sqlite.org/download.html
  * Precompiled 64-bit: https://github.com/boramalper/sqlite3-x64/releases
- R version 4.0+
  * Packages: RSQLite, tidyverse (dplyr 1.0+), jsonlite, httr
  * Precompiled versions: https://www.r-project.org/ 
    OR: https://docs.rstudio.com/resources/install-r/
- bbmap
  * https://jgi.doe.gov/data-and-tools/software-tools/bbtools/bb-tools-user-guide/installation-guide/
- sratoolkit
  * https://github.com/ncbi/sra-tools
  * !! Make sure to configure the tool before using it!!
    https://github.com/ncbi/sra-tools/wiki/03.-Quick-Toolkit-Configuration
    This needs to be done for every user who runs it (including root)
  * The default download folder is SRAdownloads/ in the SEQ2MGS folder
    This can be changed in the settings.txt file

Optional software
- pigz
  * If installed, faster zipping with multi-cores
   
IMPORTANT: Make sure all dependencies are in the $PATH variable
	HowTo: https://opensource.com/article/17/6/set-path-linux

-- END SETUP.SH ---


--- SEQ2MGS.SH ---
Mix multiple sequencing fastq.gz files together to create artificial metagenomes

See the tutorial.md file for examples on setup and running the pipeline.
https://github.com/pieterjanvc/seq2mgs/blob/master/tutorial.md

Arguments [h|i|o|l|u|a|b|d|m|t|f|v]
 -h Read the help documentation
 
# Required
 -i The input file (.csv) containing all samples to be mixed (details see below)
 -o The location to save the output file. Filename should end with fastq.gz

# Optional
 -d (optional) Change default value of the genomeSize column in the input file. 
     Default can also be changed in the settings.txt file
 -m (optional) TRUE or FALSE. 
     Generate a meta-data JSON file in the same folder as the output file.
     Default can be changed in the settings.txt file
 -f (optional) If set, force overwriting an existing output file
 -v (optional) TRUE or FALSE. Progress is posted to stdout when TRUE.
     Default can be changed in the settings.txt file
 
# File size limits for relative abundance (optional)
 -l (optional) The min number of bases the mixed file should contain
 -u (optional) The max number of bases the mixed file should contain
     
   By default, file size is defined by the number of bases in the background 
    file. If no background file is present, file size is defined as the maximum 
    number of reads that can be used from each file without resampling
	 
# Background limits for coverage (optional)
 -a (optional) The min number of bases the background should contain
 -b (optional) The max number of bases the background should contain
   
  By default, the number of bases is the difference between the
   bases used for the isolates and the bases in the original background. 
   Note that when no backgound is present, the file size is dependent on the 
   coverage settings and cannot be changed otherwise


### INPUT FILE FORMAT ###

This is a comma separated CSV file with the following columns
 - type: either I for isolate file or B for background file
   * Minimum of 2 I files if no B file
   * Max 1 B file with 1 or more I files
 - sampleName (optional): custom name for the different input files
 - genomeSize (optional): The size of each genome in bp (e.g. 3.7e6) needed to
    calculate correct genome coverage. If not set or missing values, defaults to
    value of argument -d. The value is left blank for background files (B) or 
    when working with relative abundance (column not needed)
	
 DEPENDING ON PREFERENCE EITHER
  - relativeAbundance: RA of the file in the final metagenome (0-1).
     The sum of all must be 1.0 if only isolates 
     The sum must be < 1 when there is a background (RA is calculated)
  OR
  - coverage: The times a genome should be covered
  NOTE: background files (B) can have empty values 

 DEPENDING ON PREFERENCE EITHER
 - readFile: full path to the first read file (fastq.gz format)
 - readFile2: full path to the second read file (fastq.gz format)
    Leave empty in case of 1 data file
  OR
 - getFromSRA: fill in the SRR (leave readFile/readFile2 blank)
    The file will be downloaded from SRA if not found in the 
    default download folder (location can be changed in settings.txt) 
	
 NOTE: any other columns will be ignored, but kept as meta-data

EXAMPLE CSV FILE 
```
type,sampleName,relativeAbundance,readFile,readFile2,getFromSRA
I,isolate_1,0.1,/path/isolate1_1.fastq.gz,/path/isolate1_2.fastq.gz,
I,isolate_2,0.3,,,SRR3222075
B,background,,,/path/metagenome.fastq.gz,,
```

isolate_1: RA 10%, 2 local input files
isolate_2: Grab file from SRA, RA 30%
background: RA not required, 1 local input file

More examples  can be found in the tutorial.md file or at
https://github.com/pieterjanvc/seq2mgs/blob/master/tutorial.md

-- END SEQ2MGS.SH ---