#############################################################################
   _____  _   _                              
  |  __ \| \ | |   /\                        
  | |__) |  \| |  /  \ ______ ___  ___  __ _ 
  |  _  /| . ` | / /\ \______/ __|/ _ \/ _` |
  | | \ \| |\  |/ ____ \     \__ \  __/ (_| |
  |_|  \_\_| \_/_/    \_\    |___/\___|\__, |
  Tophat2, fastqc, picard, trim galore    | |
  Samtools, bedtools, ucsc tools          |_| 
   ____  _            _ _            
  |  _ \(_)_ __   ___| (_)_ __   ___ 
  | |_) | | '_ \ / _ \ | | '_ \ / _ \
  |  __/| | |_) |  __/ | | | | |  __/
  |_|   |_| .__/ \___|_|_|_| |_|\___|
          |_|                        
 
  Institute of Cancer Sciences
  Epigenetics Unit
  University of Glasgow
  Garscube Estate
  Glasgow G61 1QH

  Report bugs to [email protected] or [email protected]

  Copyright 2016 Narendra Kumar

  This program is free software; you can redistribute it and/or modify
  it under the terms of the GNU General Public License as published by
  the Free Software Foundation; version 3 of the License.

  This program is distributed in the hope that it will be useful,
  but WITHOUT ANY WARRANTY; without even the implied warranty of
  MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the
  GNU General Public License for more details.

  You should have received a copy of the GNU General Public License
  along with this program; if not, see <http://www.gnu.org/licenses/>
  or write to the Free Software Foundation, Inc., 51 Franklin Street,
  Fifth Floor, Boston, MA 02110-1301, USA.

  Python scripts bedToBedGraph.py and extendbed were written by Tony McBryan.

  ==============================================================================



  *Prerequisite

  Install the following programs on your system

  1. Tophat2
     https://ccb.jhu.edu/software/tophat/index.shtml

  2. FASTQC
     http://www.bioinformatics.babraham.ac.uk/projects/fastqc/

  3. Picard tools
     https://broadinstitute.github.io/picard/

  4. Trim Galore
     http://www.bioinformatics.babraham.ac.uk/projects/trim_galore/

  5. SAMtools
     http://samtools.sourceforge.net/

  6. bedtools: a powerful toolset for genome arithmetic
     http://bedtools.readthedocs.io/en/latest/index.html

  7. Following programs from UCSCtools
     http://hgdownload.cse.ucsc.edu/admin/exe/linux.x86_64/ (for 64 bit linux)
     bedToBigBed
     fetchChromSizes
     wigToBigWig



  *Installation

  1. To install RNA-seq pipeline, untar the package at your favorite place.

  2. Change the value of variables in lines from 85 to 97 to the appropriate
     location of the program on your system

     Make sure you set the path to the index files of the genomes for
     bowtie2. You may download from the following locations.

     http://bowtie-bio.sourceforge.net/bowtie2/index.shtml
     http://support.illumina.com/sequencing/sequencing_software/igenome.html

  You can now start using by giving the full path of the perl script

  perl <full path to parent directory>/bin/tophat2Pipeline.pl <options>

  You may make an alias of executable as use it from anywhere without having
  to write full path

  Add following line in your ~/.bashrc file

  alias bowtie2Pipeline="<full path to parent directory>/RNAseq_Pipeline/bin/tophat2Pipeline.pl.pl"



  *User manual
  
  Usage: tophat2Pipeline.pl < --dir MAPDIRECTORY --gft GTFFILE --species SPECIES --quals QUALS > [Options]

  Options:

    -h, --help       Show this help message.

    -d, --dir        directory where sequences are kept  (required)

    -g, --gtf        gtf file (required)

    -s, --species    species e.g. hg19  (required)

    -q, --quals      quals (required)
                     available quals: phred33-quals
                                      phred64-quals
                                      solexa1.3-quals

    -t, --threads    number of processors to be used (default 2)

    -e, --extra      extra bowtie options enclosed in quote ""

    -z, --zip        zip the fastq files after the run y=yes; n=no (default y)

  Examples:

  $ perl tophat2Pipeline.pl --dir mapdirectory --gft gtffile.gtf --species hg19 --quals phred33-quals
  \or\
  $ perl tophat2Pipeline.pl  -d mapdirectory -g gtf gtffile.gtf -s hg19 -q phred33-quals -t 8 -z n

  #############################################################################
                          * End of the file *