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1 #!/usr/bin/perl --
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2 use strict;
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3 use warnings;
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4 use Cwd;
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5 use File::Path qw(rmtree);
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6 $|++;
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7
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8
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9 ## This program is Copyright (C) 2010-13, Felix Krueger (felix.krueger@babraham.ac.uk)
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10
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11 ## This program is free software: you can redistribute it and/or modify
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12 ## it under the terms of the GNU General Public License as published by
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13 ## the Free Software Foundation, either version 3 of the License, or
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14 ## (at your option) any later version.
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15
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16 ## This program is distributed in the hope that it will be useful,
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17 ## but WITHOUT ANY WARRANTY; without even the implied warranty of
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18 ## MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the
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19 ## GNU General Public License for more details.
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20
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21 ## You should have received a copy of the GNU General Public License
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22 ## along with this program. If not, see <http://www.gnu.org/licenses/>.
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23
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24 use Getopt::Long;
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25 use Cwd;
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26
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27 my $verbose;
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28 my $help;
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29 my $version;
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30 my $man;
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31 my $path_to_bowtie;
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32 my $multi_fasta;
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33 my $single_fasta;
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34 my $bowtie2;
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35
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36 my $bismark_version = 'v0.7.12';
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37
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38 GetOptions ('verbose' => \$verbose,
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39 'help' => \$help,
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40 'man' => \$man,
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41 'version' => \$version,
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42 'path_to_bowtie:s' => \$path_to_bowtie,
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43 'single_fasta' => \$single_fasta,
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44 'bowtie2' => \$bowtie2,
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45 );
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46
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47 my $genome_folder = shift @ARGV; # mandatory
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48 my $CT_dir;
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49 my $GA_dir;
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50
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51 if ($help or $man){
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52 print_helpfile();
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53 exit;
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54 }
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55
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56 if ($version){
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57 print << "VERSION";
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58
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59 Bismark - Bisulfite Mapper and Methylation Caller.
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60
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61 Bismark Genome Preparation Version: $bismark_version
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62 Copyright 2010-13 Felix Krueger, Babraham Bioinformatics
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63 www.bioinformatics.babraham.ac.uk/projects/
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64
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65 VERSION
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66 exit;
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67 }
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68
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69 if ($single_fasta){
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70 print "Writing individual genomes out into single-entry fasta files (one per chromosome)\n\n";
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71 $multi_fasta = 0;
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72 }
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73 else{
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74 print "Writing bisulfite genomes out into a single MFA (multi FastA) file\n\n";
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75 $single_fasta = 0;
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76 $multi_fasta = 1;
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77 }
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78
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79 my @filenames = create_bisulfite_genome_folders();
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80
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81 process_sequence_files ();
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82
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83 launch_bowtie_indexer();
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84
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85 sub launch_bowtie_indexer{
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86 if ($bowtie2){
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87 print "Bismark Genome Preparation - Step III: Launching the Bowtie 2 indexer\n";
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88 }
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89 else{
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90 print "Bismark Genome Preparation - Step III: Launching the Bowtie (1) indexer\n";
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91 }
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92 print "Please be aware that this process can - depending on genome size - take up to several hours!\n";
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93 sleep(5);
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94
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95 ### if the path to bowtie was specfified explicitely
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96 if ($path_to_bowtie){
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97 if ($bowtie2){
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98 $path_to_bowtie =~ s/$/bowtie2-build/;
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99 }
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100 else{
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101 $path_to_bowtie =~ s/$/bowtie-build/;
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102 }
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103 }
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104 ### otherwise we assume that bowtie-build is in the path
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105 else{
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106 if ($bowtie2){
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107 $path_to_bowtie = 'bowtie2-build';
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108 }
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109 else{
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110 $path_to_bowtie = 'bowtie-build';
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111 }
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112 }
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113
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114 $verbose and print "\n";
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115
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116 ### Forking the program to run 2 instances of Bowtie-build or Bowtie2-build (= the Bowtie (1/2) indexer)
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117 my $pid = fork();
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118
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119 # parent process
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120 if ($pid){
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121 sleep(1);
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122 chdir $CT_dir or die "Unable to change directory: $!\n";
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123 $verbose and warn "Preparing indexing of CT converted genome in $CT_dir\n";
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124 my @fasta_files = <*.fa>;
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125 my $file_list = join (',',@fasta_files);
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126 $verbose and print "Parent process: Starting to index C->T converted genome with the following command:\n\n";
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127 $verbose and print "$path_to_bowtie -f $file_list BS_CT\n\n";
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128
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129 sleep (11);
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130 exec ("$path_to_bowtie","-f","$file_list","BS_CT");
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131 }
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132
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133 # child process
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134 elsif ($pid == 0){
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135 sleep(2);
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136 chdir $GA_dir or die "Unable to change directory: $!\n";
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137 $verbose and warn "Preparing indexing of GA converted genome in $GA_dir\n";
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138 my @fasta_files = <*.fa>;
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139 my $file_list = join (',',@fasta_files);
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140 $verbose and print "Child process: Starting to index G->A converted genome with the following command:\n\n";
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141 $verbose and print "$path_to_bowtie -f $file_list BS_GA\n\n";
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142 $verbose and print "(starting in 10 seconds)\n";
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143 sleep(10);
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144 exec ("$path_to_bowtie","-f","$file_list","BS_GA");
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145 }
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146
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147 # if the platform doesn't support the fork command we will run the indexing processes one after the other
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148 else{
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149 print "Forking process was not successful, therefore performing the indexing sequentially instead\n";
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150 sleep(10);
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151
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152 ### moving to CT genome folder
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153 $verbose and warn "Preparing to index CT converted genome in $CT_dir\n";
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154 chdir $CT_dir or die "Unable to change directory: $!\n";
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155 my @fasta_files = <*.fa>;
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156 my $file_list = join (',',@fasta_files);
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157 $verbose and print "$file_list\n\n";
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158 sleep(2);
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159 system ("$path_to_bowtie","-f","$file_list","BS_CT");
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160 @fasta_files=();
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161 $file_list= '';
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162
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163 ### moving to GA genome folder
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164 $verbose and warn "Preparing to index GA converted genome in $GA_dir\n";
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165 chdir $GA_dir or die "Unable to change directory: $!\n";
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166 @fasta_files = <*.fa>;
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167 $file_list = join (',',@fasta_files);
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168 $verbose and print "$file_list\n\n";
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169 sleep(2);
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170 exec ("$path_to_bowtie","-f","$file_list","BS_GA");
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171 }
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172 }
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173
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174
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175 sub process_sequence_files {
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176
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177 my ($total_CT_conversions,$total_GA_conversions) = (0,0);
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178 $verbose and print "Bismark Genome Preparation - Step II: Bisulfite converting reference genome\n\n";
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179 sleep (3);
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180
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181 $verbose and print "conversions performed:\n";
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182 $verbose and print join("\t",'chromosome','C->T','G->A'),"\n";
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183
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184
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185 ### If someone wants to index a genome which consists of thousands of contig and scaffold files we need to write the genome conversions into an MFA file
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186 ### Otherwise the list of comma separated chromosomes we provide for bowtie-build will get too long for the kernel to handle
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187 ### This is now the default option
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188
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189 if ($multi_fasta){
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190 ### Here we just use one multi FastA file name, append .CT_conversion or .GA_conversion and print all sequence conversions into these files
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191 my $bisulfite_CT_conversion_filename = "$CT_dir/genome_mfa.CT_conversion.fa";
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192 open (CT_CONVERT,'>',$bisulfite_CT_conversion_filename) or die "Can't write to file $bisulfite_CT_conversion_filename: $!\n";
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193
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194 my $bisulfite_GA_conversion_filename = "$GA_dir/genome_mfa.GA_conversion.fa";
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195 open (GA_CONVERT,'>',$bisulfite_GA_conversion_filename) or die "Can't write to file $bisulfite_GA_conversion_filename: $!\n";
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196 }
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197
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198 foreach my $filename(@filenames){
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199 my ($chromosome_CT_conversions,$chromosome_GA_conversions) = (0,0);
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200 open (IN,$filename) or die "Failed to read from sequence file $filename $!\n";
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201 # warn "Reading chromosome information from $filename\n\n";
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202
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203 ### first line needs to be a fastA header
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204 my $first_line = <IN>;
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205 chomp $first_line;
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206
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207 ### Extracting chromosome name from the FastA header
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208 my $chromosome_name = extract_chromosome_name($first_line);
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209
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210 ### alternatively, chromosomes can be written out into single-entry FastA files. This will only work for genomes with up to a few hundred chromosomes.
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211 unless ($multi_fasta){
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212 my $bisulfite_CT_conversion_filename = "$CT_dir/$chromosome_name";
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213 $bisulfite_CT_conversion_filename =~ s/$/.CT_conversion.fa/;
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214 open (CT_CONVERT,'>',$bisulfite_CT_conversion_filename) or die "Can't write to file $bisulfite_CT_conversion_filename: $!\n";
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215
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216 my $bisulfite_GA_conversion_filename = "$GA_dir/$chromosome_name";
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217 $bisulfite_GA_conversion_filename =~ s/$/.GA_conversion.fa/;
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218 open (GA_CONVERT,'>',$bisulfite_GA_conversion_filename) or die "Can't write to file $bisulfite_GA_conversion_filename: $!\n";
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219 }
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220
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221 print CT_CONVERT ">",$chromosome_name,"_CT_converted\n"; # first entry
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222 print GA_CONVERT ">",$chromosome_name,"_GA_converted\n"; # first entry
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223
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224
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225 while (<IN>){
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226
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227 ### in case the line is a new fastA header
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228 if ($_ =~ /^>/){
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229 ### printing out the stats for the previous chromosome
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230 $verbose and print join ("\t",$chromosome_name,$chromosome_CT_conversions,$chromosome_GA_conversions),"\n";
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231 ### resetting the chromosome transliteration counters
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232 ($chromosome_CT_conversions,$chromosome_GA_conversions) = (0,0);
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233
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234 ### Extracting chromosome name from the additional FastA header
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235 $chromosome_name = extract_chromosome_name($_);
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236
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237 ### alternatively, chromosomes can be written out into single-entry FastA files. This will only work for genomes with up to a few hundred chromosomes.
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238 unless ($multi_fasta){
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239 my $bisulfite_CT_conversion_filename = "$CT_dir/$chromosome_name";
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240 $bisulfite_CT_conversion_filename =~ s/$/.CT_conversion.fa/;
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241 open (CT_CONVERT,'>',$bisulfite_CT_conversion_filename) or die "Can't write to file $bisulfite_CT_conversion_filename: $!\n";
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242
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243 my $bisulfite_GA_conversion_filename = "$GA_dir/$chromosome_name";
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244 $bisulfite_GA_conversion_filename =~ s/$/.GA_conversion.fa/;
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245 open (GA_CONVERT,'>',$bisulfite_GA_conversion_filename) or die "Can't write to file $bisulfite_GA_conversion_filename: $!\n";
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246 }
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247
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248 print CT_CONVERT ">",$chromosome_name,"_CT_converted\n";
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249 print GA_CONVERT ">",$chromosome_name,"_GA_converted\n";
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250 }
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251
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252 else{
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253 my $sequence = uc$_;
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254
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255 ### (I) First replacing all ambiguous sequence characters (such as M,S,R....) by N (G,A,T,C,N and the line endings \r and \n are added to a character group)
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256
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257 $sequence =~ s/[^ATCGN\n\r]/N/g;
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258
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259 ### (II) Writing the chromosome out into a C->T converted version (equals forward strand conversion)
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260
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261 my $CT_sequence = $sequence;
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262 my $CT_transliterations_performed = ($CT_sequence =~ tr/C/T/); # converts all Cs into Ts
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263 $total_CT_conversions += $CT_transliterations_performed;
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264 $chromosome_CT_conversions += $CT_transliterations_performed;
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265
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266 print CT_CONVERT $CT_sequence;
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267
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268 ### (III) Writing the chromosome out in a G->A converted version of the forward strand (this is equivalent to reverse-
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269 ### complementing the forward strand and then C->T converting it)
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270
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271 my $GA_sequence = $sequence;
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272 my $GA_transliterations_performed = ($GA_sequence =~ tr/G/A/); # converts all Gs to As on the forward strand
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273 $total_GA_conversions += $GA_transliterations_performed;
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274 $chromosome_GA_conversions += $GA_transliterations_performed;
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275
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276 print GA_CONVERT $GA_sequence;
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277
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278 }
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279 }
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280 $verbose and print join ("\t",$chromosome_name,$chromosome_CT_conversions,$chromosome_GA_conversions),"\n";
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281 }
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282 close (CT_CONVERT) or die "Failed to close filehandle: $!\n";
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283 close (GA_CONVERT) or die "Failed to close filehandle: $!\n";
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284
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285
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286 print "\nTotal number of conversions performed:\n";
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287 print "C->T:\t$total_CT_conversions\n";
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288 print "G->A:\t$total_GA_conversions\n";
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289
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290 warn "\nStep II - Genome bisulfite conversions - completed\n\n\n";
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291 }
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292
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293 sub extract_chromosome_name {
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294
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295 my $header = shift;
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296
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297 ## Bowtie extracts the first string after the initial > in the FASTA file, so we are doing this as well
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298
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299 if ($header =~ s/^>//){
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300 my ($chromosome_name) = split (/\s+/,$header);
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301 return $chromosome_name;
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302 }
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303 else{
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304 die "The specified chromosome file doesn't seem to be in FASTA format as required! $!\n";
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305 }
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306 }
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307
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308 sub create_bisulfite_genome_folders{
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309
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310 $verbose and print "Bismark Genome Preparation - Step I: Preparing folders\n\n";
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311
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312 # Ensuring a genome folder has been specified
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313 if ($genome_folder){
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314 unless ($genome_folder =~ /\/$/){
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315 $genome_folder =~ s/$/\//;
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316 }
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317 $verbose and print "Path to genome folder specified: $genome_folder\n";
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318 chdir $genome_folder or die "Could't move to directory $genome_folder. Make sure the directory exists! $!";
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319
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320 # making the genome folder path abolsolute so it won't break if the path was specified relative
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321 $genome_folder = getcwd;
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322 unless ($genome_folder =~ /\/$/){
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323 $genome_folder =~ s/$/\//;
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324 }
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325 }
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326
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327 else{
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328 $verbose and print "Genome folder was not provided as argument ";
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329 while (1){
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330 print "Please specify a genome folder to be bisulfite converted:\n";
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331 $genome_folder = <STDIN>;
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332 chomp $genome_folder;
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333
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334 # adding a trailing slash unless already present
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335 unless ($genome_folder =~ /\/$/){
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336 $genome_folder =~ s/$/\//;
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337 }
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338 if (chdir $genome_folder){
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339 last;
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340 }
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341 else{
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342 warn "Could't move to directory $genome_folder! $!";
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343 }
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344 }
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345 }
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346
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347 if ($path_to_bowtie){
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348 unless ($path_to_bowtie =~ /\/$/){
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349 $path_to_bowtie =~ s/$/\//;
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350 }
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351 if (chdir $path_to_bowtie){
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352 if ($bowtie2){
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353 $verbose and print "Path to Bowtie 2 specified: $path_to_bowtie\n";
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354 }
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355 else{
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356 $verbose and print "Path to Bowtie (1) specified: $path_to_bowtie\n";
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357 }
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358 }
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359 else{
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360 die "There was an error with the path to bowtie: $!\n";
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361 }
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362 }
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363
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364 chdir $genome_folder or die "Could't move to directory $genome_folder. Make sure the directory exists! $!";
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365
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366
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367 # Exiting unless there are fastA files in the folder
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368 my @filenames = <*.fa>;
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369
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370 ### if there aren't any genomic files with the extension .fa we will look for files with the extension .fasta
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371 unless (@filenames){
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372 @filenames = <*.fasta>;
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373 }
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374
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375 unless (@filenames){
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376 die "The specified genome folder $genome_folder does not contain any sequence files in FastA format (with .fa or .fasta file extensions\n";
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377 }
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378
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379 warn "Bisulfite Genome Indexer version $bismark_version (last modified 17 Nov 2011)\n\n";
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380 sleep (3);
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381
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382 # creating a directory inside the genome folder to store the bisfulfite genomes unless it already exists
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383 my $bisulfite_dir = "${genome_folder}Bisulfite_Genome/";
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384 unless (-d $bisulfite_dir){
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385 mkdir $bisulfite_dir or die "Unable to create directory $bisulfite_dir $!\n";
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386 $verbose and print "Created Bisulfite Genome folder $bisulfite_dir\n";
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387 }
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388 else{
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389 while (1){
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390 print "\nA directory called $bisulfite_dir already exists. Bisulfite converted sequences and/or already existing Bowtie (1 or 2) indexes might be overwritten!\nDo you want to continue anyway?\t";
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391 my $proceed = <STDIN>;
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392 chomp $proceed;
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393 if ($proceed =~ /^y/i ){
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394 last;
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395 }
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396 elsif ($proceed =~ /^n/i){
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397 die "Terminated by user\n\n";
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398 }
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399 }
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400 }
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401
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402 ### as of version 0.6.0 the Bismark indexer will no longer delete the Bisulfite_Genome directory if it was present already, since it could store the Bowtie 1 or 2 indexes already
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403 # removing any existing files and subfolders in the bisulfite directory (the specified directory won't be deleted)
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404 # rmtree($bisulfite_dir, {verbose => 1,keep_root => 1});
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405 # unless (-d $bisulfite_dir){ # had to add this after changing remove_tree to rmtree // suggested by Samantha Cooper @ Illumina
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406 # mkdir $bisulfite_dir or die "Unable to create directory $bisulfite_dir $!\n";
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407 # }
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408 # }
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409
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410 chdir $bisulfite_dir or die "Unable to move to $bisulfite_dir\n";
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411 $CT_dir = "${bisulfite_dir}CT_conversion/";
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412 $GA_dir = "${bisulfite_dir}GA_conversion/";
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413
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414 # creating 2 subdirectories to store a C->T (forward strand conversion) and a G->A (reverse strand conversion)
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415 # converted version of the genome
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416 unless (-d $CT_dir){
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417 mkdir $CT_dir or die "Unable to create directory $CT_dir $!\n";
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418 $verbose and print "Created Bisulfite Genome folder $CT_dir\n";
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419 }
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420 unless (-d $GA_dir){
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421 mkdir $GA_dir or die "Unable to create directory $GA_dir $!\n";
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422 $verbose and print "Created Bisulfite Genome folder $GA_dir\n";
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423 }
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424
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425 # moving back to the original genome folder
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426 chdir $genome_folder or die "Could't move to directory $genome_folder $!";
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427 # $verbose and print "Moved back to genome folder folder $genome_folder\n";
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428 warn "\nStep I - Prepare genome folders - completed\n\n\n";
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429 return @filenames;
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430 }
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431
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432 sub print_helpfile{
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433 print << 'HOW_TO';
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434
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435
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436 DESCRIPTION
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437
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438 This script is supposed to convert a specified reference genome into two different bisulfite
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439 converted versions and index them for alignments with Bowtie 1 (default), or Bowtie 2. The first
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440 bisulfite genome will have all Cs converted to Ts (C->T), and the other one will have all Gs
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441 converted to As (G->A). Both bisulfite genomes will be stored in subfolders within the reference
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442 genome folder. Once the bisulfite conversion has been completed the program will fork and launch
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443 two simultaneous instances of the bowtie 1 or 2 indexer (bowtie-build or bowtie2-build). Be aware
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444 that the indexing process can take up to several hours; this will mainly depend on genome size
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445 and system resources.
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446
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447
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448
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449
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450 The following is a brief description of command line options and arguments to control the
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|
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451 Bismark Genome Preparation script:
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452
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453
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454 USAGE: bismark_genome_preparation [options] <arguments>
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455
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456
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457 OPTIONS:
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458
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459 --help/--man Displays this help filea and exits.
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460
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461 --version Displays version information and exits.
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462
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463 --verbose Print verbose output for more details or debugging.
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464
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465 --path_to_bowtie The full path to the Bowtie 1 or Bowtie 2 installation on your system.If
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466 the path </../../> is not provided as an option you will be prompted for it.
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467
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468 --bowtie2 This will create bisulfite indexes for Bowtie 2. (Default: Bowtie 1).
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469
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470 --single_fasta Instruct the Bismark Indexer to write the converted genomes into
|
|
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471 single-entry FastA files instead of making one multi-FastA file (MFA)
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|
|
472 per chromosome. This might be useful if individual bisulfite converted
|
|
|
473 chromosomes are needed (e.g. for debugging), however it can cause a
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474 problem with indexing if the number of chromosomes is vast (this is likely
|
|
|
475 to be in the range of several thousand files; the operating system can
|
|
|
476 only handle lists up to a certain length, and some newly assembled
|
|
|
477 genomes may contain 20000-50000 contigs of scaffold files which do exceed
|
|
|
478 this list length limit).
|
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479
|
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480
|
|
|
481 ARGUMENTS:
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482
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483 <path_to_genome_folder> The path to the folder containing the genome to be bisulfite converted.
|
|
|
484 At the current time Bismark Genome Preparation expects one or more fastA
|
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485 files in the folder (with the file extension: .fa or .fasta). If the path
|
|
|
486 is not provided as an argument you will be prompted for it.
|
|
|
487
|
|
|
488
|
|
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489
|
|
|
490 This script was last modified on 18 Nov 2011.
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|
|
491 HOW_TO
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|
|
492 }
|
