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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 IO::Handle;
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5 use Cwd;
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6 $|++;
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7 use Getopt::Long;
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8
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9
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10 ## This program is Copyright (C) 2010-12, Felix Krueger (felix.krueger@babraham.ac.uk)
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11
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12 ## This program is free software: you can redistribute it and/or modify
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13 ## it under the terms of the GNU General Public License as published by
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14 ## the Free Software Foundation, either version 3 of the License, or
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15 ## (at your option) any later version.
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16
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17 ## This program is distributed in the hope that it will be useful,
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18 ## but WITHOUT ANY WARRANTY; without even the implied warranty of
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19 ## MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the
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20 ## GNU General Public License for more details.
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21
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22 ## You should have received a copy of the GNU General Public License
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23 ## along with this program. If not, see <http://www.gnu.org/licenses/>.
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24
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25
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26 my $parent_dir = getcwd;
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27 my $bismark_version = 'v0.7.7';
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28 my $command_line = join (" ",@ARGV);
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29
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30 ### before processing the command line we will replace --solexa1.3-quals with --phred64-quals as the '.' in the option name will cause Getopt::Long to fail
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31 foreach my $arg (@ARGV){
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32 if ($arg eq '--solexa1.3-quals'){
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33 $arg = '--phred64-quals';
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34 }
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35 }
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36 my @filenames; # will be populated by processing the command line
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37
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38 my ($genome_folder,$CT_index_basename,$GA_index_basename,$path_to_bowtie,$sequence_file_format,$bowtie_options,$directional,$unmapped,$ambiguous,$phred64,$solexa,$output_dir,$bowtie2,$vanilla,$sam_no_hd,$skip,$upto,$temp_dir) = process_command_line();
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39
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40 my @fhs; # stores alignment process names, bisulfite index location, bowtie filehandles and the number of times sequences produced an alignment
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41 my %chromosomes; # stores the chromosome sequences of the mouse genome
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42 my %counting; # counting various events
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43
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44 my $seqID_contains_tabs;
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45
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46 foreach my $filename (@filenames){
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47
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48 chdir $parent_dir or die "Unable to move to initial working directory $!\n";
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49 ### resetting the counting hash and fhs
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50 reset_counters_and_fhs($filename);
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51 $seqID_contains_tabs = 0;
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52
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53 ### PAIRED-END ALIGNMENTS
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54 if ($filename =~ ','){
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55 my ($C_to_T_infile_1,$G_to_A_infile_1); # to be made from mate1 file
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56
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57 $fhs[0]->{name} = 'CTread1GAread2CTgenome';
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58 $fhs[1]->{name} = 'GAread1CTread2GAgenome';
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59 $fhs[2]->{name} = 'GAread1CTread2CTgenome';
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60 $fhs[3]->{name} = 'CTread1GAread2GAgenome';
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61
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62 print "\nPaired-end alignments will be performed\n",'='x39,"\n\n";
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63
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64 my ($filename_1,$filename_2) = (split (/,/,$filename));
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65 print "The provided filenames for paired-end alignments are $filename_1 and $filename_2\n";
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66
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67 ### additional variables only for paired-end alignments
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68 my ($C_to_T_infile_2,$G_to_A_infile_2); # to be made from mate2 file
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69
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70 ### FastA format
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71 if ($sequence_file_format eq 'FASTA'){
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72 print "Input files are in FastA format\n";
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73
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74 if ($directional){
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75 ($C_to_T_infile_1) = biTransformFastAFiles_paired_end ($filename_1,1); # also passing the read number
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76 ($G_to_A_infile_2) = biTransformFastAFiles_paired_end ($filename_2,2);
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77
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78 $fhs[0]->{inputfile_1} = $C_to_T_infile_1;
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79 $fhs[0]->{inputfile_2} = $G_to_A_infile_2;
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80 $fhs[1]->{inputfile_1} = undef;
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81 $fhs[1]->{inputfile_2} = undef;
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82 $fhs[2]->{inputfile_1} = undef;
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83 $fhs[2]->{inputfile_2} = undef;
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84 $fhs[3]->{inputfile_1} = $C_to_T_infile_1;
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85 $fhs[3]->{inputfile_2} = $G_to_A_infile_2;
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86 }
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87 else{
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88 ($C_to_T_infile_1,$G_to_A_infile_1) = biTransformFastAFiles_paired_end ($filename_1,1); # also passing the read number
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89 ($C_to_T_infile_2,$G_to_A_infile_2) = biTransformFastAFiles_paired_end ($filename_2,2);
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90
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91 $fhs[0]->{inputfile_1} = $C_to_T_infile_1;
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92 $fhs[0]->{inputfile_2} = $G_to_A_infile_2;
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93 $fhs[1]->{inputfile_1} = $G_to_A_infile_1;
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94 $fhs[1]->{inputfile_2} = $C_to_T_infile_2;
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95 $fhs[2]->{inputfile_1} = $G_to_A_infile_1;
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96 $fhs[2]->{inputfile_2} = $C_to_T_infile_2;
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97 $fhs[3]->{inputfile_1} = $C_to_T_infile_1;
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98 $fhs[3]->{inputfile_2} = $G_to_A_infile_2;
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99 }
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100
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101 if ($bowtie2){
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102 paired_end_align_fragments_to_bisulfite_genome_fastA_bowtie2 ($C_to_T_infile_1,$G_to_A_infile_1,$C_to_T_infile_2,$G_to_A_infile_2);
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103 }
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104 else{
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105 paired_end_align_fragments_to_bisulfite_genome_fastA ($C_to_T_infile_1,$G_to_A_infile_1,$C_to_T_infile_2,$G_to_A_infile_2);
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106 }
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107 }
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108
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109 ### FastQ format
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110 else{
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111 print "Input files are in FastQ format\n";
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112 if ($directional){
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113 ($C_to_T_infile_1) = biTransformFastQFiles_paired_end ($filename_1,1); # also passing the read number
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114 ($G_to_A_infile_2) = biTransformFastQFiles_paired_end ($filename_2,2);
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115
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116 $fhs[0]->{inputfile_1} = $C_to_T_infile_1;
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117 $fhs[0]->{inputfile_2} = $G_to_A_infile_2;
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118 $fhs[1]->{inputfile_1} = undef;
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119 $fhs[1]->{inputfile_2} = undef;
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120 $fhs[2]->{inputfile_1} = undef;
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121 $fhs[2]->{inputfile_2} = undef;
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122 $fhs[3]->{inputfile_1} = $C_to_T_infile_1;
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123 $fhs[3]->{inputfile_2} = $G_to_A_infile_2;
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124 }
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125 else{
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126 ($C_to_T_infile_1,$G_to_A_infile_1) = biTransformFastQFiles_paired_end ($filename_1,1); # also passing the read number
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127 ($C_to_T_infile_2,$G_to_A_infile_2) = biTransformFastQFiles_paired_end ($filename_2,2);
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128
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129 $fhs[0]->{inputfile_1} = $C_to_T_infile_1;
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130 $fhs[0]->{inputfile_2} = $G_to_A_infile_2;
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131 $fhs[1]->{inputfile_1} = $G_to_A_infile_1;
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132 $fhs[1]->{inputfile_2} = $C_to_T_infile_2;
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133 $fhs[2]->{inputfile_1} = $G_to_A_infile_1;
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134 $fhs[2]->{inputfile_2} = $C_to_T_infile_2;
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135 $fhs[3]->{inputfile_1} = $C_to_T_infile_1;
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136 $fhs[3]->{inputfile_2} = $G_to_A_infile_2;
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137 }
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138
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139 if ($bowtie2){
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140 paired_end_align_fragments_to_bisulfite_genome_fastQ_bowtie2 ($C_to_T_infile_1,$G_to_A_infile_1,$C_to_T_infile_2,$G_to_A_infile_2);
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141 }
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142 else{
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143 paired_end_align_fragments_to_bisulfite_genome_fastQ ($C_to_T_infile_1,$G_to_A_infile_1,$C_to_T_infile_2,$G_to_A_infile_2);
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144 }
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145 }
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146 start_methylation_call_procedure_paired_ends($filename_1,$filename_2,$C_to_T_infile_1,$G_to_A_infile_1,$C_to_T_infile_2,$G_to_A_infile_2);
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147 }
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148
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149 ### Else we are performing SINGLE-END ALIGNMENTS
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150 else{
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151 print "\nSingle-end alignments will be performed\n",'='x39,"\n\n";
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152 ### Initialising bisulfite conversion filenames
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153 my ($C_to_T_infile,$G_to_A_infile);
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154
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155
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156 ### FastA format
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157 if ($sequence_file_format eq 'FASTA'){
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158 print "Inut file is in FastA format\n";
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159 if ($directional){
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160 ($C_to_T_infile) = biTransformFastAFiles ($filename);
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161 $fhs[0]->{inputfile} = $fhs[1]->{inputfile} = $C_to_T_infile;
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162 }
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163 else{
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164 ($C_to_T_infile,$G_to_A_infile) = biTransformFastAFiles ($filename);
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165 $fhs[0]->{inputfile} = $fhs[1]->{inputfile} = $C_to_T_infile;
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166 $fhs[2]->{inputfile} = $fhs[3]->{inputfile} = $G_to_A_infile;
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167 }
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168
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169 ### Creating 4 different bowtie filehandles and storing the first entry
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170 if ($bowtie2){
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171 single_end_align_fragments_to_bisulfite_genome_fastA_bowtie2 ($C_to_T_infile,$G_to_A_infile);
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172 }
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173 else{
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174 single_end_align_fragments_to_bisulfite_genome_fastA ($C_to_T_infile,$G_to_A_infile);
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175 }
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176 }
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177
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178 ## FastQ format
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179 else{
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180 print "Input file is in FastQ format\n";
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181 if ($directional){
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182 ($C_to_T_infile) = biTransformFastQFiles ($filename);
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183 $fhs[0]->{inputfile} = $fhs[1]->{inputfile} = $C_to_T_infile;
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184 }
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185 else{
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186 ($C_to_T_infile,$G_to_A_infile) = biTransformFastQFiles ($filename);
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187 $fhs[0]->{inputfile} = $fhs[1]->{inputfile} = $C_to_T_infile;
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188 $fhs[2]->{inputfile} = $fhs[3]->{inputfile} = $G_to_A_infile;
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189 }
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190
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191 ### Creating 4 different bowtie filehandles and storing the first entry
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192 if ($bowtie2){
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193 single_end_align_fragments_to_bisulfite_genome_fastQ_bowtie2 ($C_to_T_infile,$G_to_A_infile);
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194 }
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195 else{
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196 single_end_align_fragments_to_bisulfite_genome_fastQ ($C_to_T_infile,$G_to_A_infile);
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197 }
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198 }
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199
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200 start_methylation_call_procedure_single_ends($filename,$C_to_T_infile,$G_to_A_infile);
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201
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202 }
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203 }
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204
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205 sub start_methylation_call_procedure_single_ends {
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206 my ($sequence_file,$C_to_T_infile,$G_to_A_infile) = @_;
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207 my ($dir,$filename);
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208
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209 if ($sequence_file =~ /\//){
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210 ($dir,$filename) = $sequence_file =~ m/(.*\/)(.*)$/;
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211 }
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212 else{
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213 $filename = $sequence_file;
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214 }
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215
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216 ### printing all alignments to a results file
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217 my $outfile = $filename;
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218
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219 if ($bowtie2){ # SAM format is the default for Bowtie 2
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220 $outfile =~ s/$/_bt2_bismark.sam/;
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221 }
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222 elsif ($vanilla){ # vanilla custom Bismark output single-end output (like Bismark versions 0.5.X)
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223 $outfile =~ s/$/_bismark.txt/;
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224 }
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225 else{ # SAM is the default output
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226 $outfile =~ s/$/_bismark.sam/;
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227 }
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228 print "Writing bisulfite mapping results to $output_dir$outfile\n\n";
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229 open (OUT,'>',"$output_dir$outfile") or die "Failed to write to $outfile: $!\n";
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230 if ($vanilla){
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231 print OUT "Bismark version: $bismark_version\n";
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232 }
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233
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234 ### printing alignment and methylation call summary to a report file
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235 my $reportfile = $filename;
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236 if ($bowtie2){
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237 $reportfile =~ s/$/_bt2_Bismark_mapping_report.txt/;
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238 }
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239 else{
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240 $reportfile =~ s/$/_Bismark_mapping_report.txt/;
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241 }
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242
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243 open (REPORT,'>',"$output_dir$reportfile") or die "Failed to write to $reportfile: $!\n";
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244 print REPORT "Bismark report for: $sequence_file (version: $bismark_version)\n";
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245
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246 if ($unmapped){
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247 my $unmapped_file = $filename;
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248 $unmapped_file =~ s/$/_unmapped_reads.txt/;
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249 open (UNMAPPED,'>',"$output_dir$unmapped_file") or die "Failed to write to $unmapped_file: $!\n";
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250 print "Unmapped sequences will be written to $output_dir$unmapped_file\n";
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251 }
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252 if ($ambiguous){
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253 my $ambiguous_file = $filename;
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254 $ambiguous_file =~ s/$/_ambiguous_reads.txt/;
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255 open (AMBIG,'>',"$output_dir$ambiguous_file") or die "Failed to write to $ambiguous_file: $!\n";
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256 print "Ambiguously mapping sequences will be written to $output_dir$ambiguous_file\n";
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257 }
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258
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259 if ($directional){
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260 print REPORT "Option '--directional' specified: alignments to complementary strands will be ignored (i.e. not performed!)\n";
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261 }
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262 print REPORT "Bowtie was run against the bisulfite genome of $genome_folder with the specified options: $bowtie_options\n\n";
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263
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264
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265 ### if 2 or more files are provided we can hold the genome in memory and don't need to read it in a second time
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266 unless (%chromosomes){
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267 my $cwd = getcwd; # storing the path of the current working directory
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268 print "Current working directory is: $cwd\n\n";
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269 read_genome_into_memory($cwd);
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270 }
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271
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272 unless ($vanilla or $sam_no_hd){
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273 generate_SAM_header();
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274 }
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275
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276 ### Input file is in FastA format
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277 if ($sequence_file_format eq 'FASTA'){
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278 process_single_end_fastA_file_for_methylation_call($sequence_file,$C_to_T_infile,$G_to_A_infile);
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279 }
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280 ### Input file is in FastQ format
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281 else{
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282 process_single_end_fastQ_file_for_methylation_call($sequence_file,$C_to_T_infile,$G_to_A_infile);
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283 }
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284 }
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285
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286 sub start_methylation_call_procedure_paired_ends {
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287 my ($sequence_file_1,$sequence_file_2,$C_to_T_infile_1,$G_to_A_infile_1,$C_to_T_infile_2,$G_to_A_infile_2) = @_;
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288
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289 my ($dir_1,$filename_1);
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290
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291 if ($sequence_file_1 =~ /\//){
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292 ($dir_1,$filename_1) = $sequence_file_1 =~ m/(.*\/)(.*)$/;
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293 }
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294 else{
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295 $filename_1 = $sequence_file_1;
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296 }
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297
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298 my ($dir_2,$filename_2);
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299
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300 if ($sequence_file_2 =~ /\//){
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301 ($dir_2,$filename_2) = $sequence_file_2 =~ m/(.*\/)(.*)$/;
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302 }
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303 else{
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304 $filename_2 = $sequence_file_2;
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305 }
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306
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307 ### printing all alignments to a results file
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308 my $outfile = $filename_1;
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309 if ($bowtie2){ # SAM format is the default Bowtie 2 output
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310 $outfile =~ s/$/_bismark_bt2_pe.sam/;
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311 }
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312 elsif ($vanilla){ # vanilla custom Bismark paired-end output (like Bismark versions 0.5.X)
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313 $outfile =~ s/$/_bismark_pe.txt/;
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314 }
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315 else{ # SAM format is the default Bowtie 1 output
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316 $outfile =~ s/$/_bismark_pe.sam/;
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317 }
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318
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319 print "Writing bisulfite mapping results to $outfile\n\n";
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320 open (OUT,'>',"$output_dir$outfile") or die "Failed to write to $outfile: $!";
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321 if ($vanilla){
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322 print OUT "Bismark version: $bismark_version\n";
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323 }
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324
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325 ### printing alignment and methylation call summary to a report file
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326 my $reportfile = $filename_1;
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327 if ($bowtie2){
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328 $reportfile =~ s/$/_Bismark_bt2_paired-end_mapping_report.txt/;
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329 }
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330 else{
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331 $reportfile =~ s/$/_Bismark_paired-end_mapping_report.txt/;
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332 }
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333
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334 open (REPORT,'>',"$output_dir$reportfile") or die "Failed to write to $reportfile: $!\n";
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335 print REPORT "Bismark report for: $sequence_file_1 and $sequence_file_2 (version: $bismark_version)\n";
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336 print REPORT "Bowtie was run against the bisulfite genome of $genome_folder with the specified options: $bowtie_options\n\n";
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337
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338
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339 ### Unmapped read output
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340 if ($unmapped){
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341 my $unmapped_1 = $filename_1;
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342 my $unmapped_2 = $filename_2;
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343 $unmapped_1 =~ s/$/_unmapped_reads_1.txt/;
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344 $unmapped_2 =~ s/$/_unmapped_reads_2.txt/;
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345 open (UNMAPPED_1,'>',"$output_dir$unmapped_1") or die "Failed to write to $unmapped_1: $!\n";
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346 open (UNMAPPED_2,'>',"$output_dir$unmapped_2") or die "Failed to write to $unmapped_2: $!\n";
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347 print "Unmapped sequences will be written to $unmapped_1 and $unmapped_2\n";
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348 }
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349
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350 if ($ambiguous){
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351 my $amb_1 = $filename_1;
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352 my $amb_2 = $filename_2;
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353 $amb_1 =~ s/$/_ambiguous_reads_1.txt/;
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354 $amb_2 =~ s/$/_ambiguous_reads_2.txt/;
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355 open (AMBIG_1,'>',"$output_dir$amb_1") or die "Failed to write to $amb_1: $!\n";
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356 open (AMBIG_2,'>',"$output_dir$amb_2") or die "Failed to write to $amb_2: $!\n";
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357 print "Ambiguously mapping sequences will be written to $amb_1 and $amb_2\n";
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358 }
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359
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360 if ($directional){
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361 print REPORT "Option '--directional' specified: alignments to complementary strands will be ignored (i.e. not performed)\n";
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362 }
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363
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364 ### if 2 or more files are provided we might still hold the genome in memory and don't need to read it in a second time
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365 unless (%chromosomes){
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366 my $cwd = getcwd; # storing the path of the current working directory
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367 print "Current working directory is: $cwd\n\n";
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368 read_genome_into_memory($cwd);
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369 }
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370
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371 unless ($vanilla or $sam_no_hd){
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372 generate_SAM_header();
|
|
373 }
|
|
374
|
|
375 ### Input files are in FastA format
|
|
376 if ($sequence_file_format eq 'FASTA'){
|
|
377 process_fastA_files_for_paired_end_methylation_calls($sequence_file_1,$sequence_file_2,$C_to_T_infile_1,$G_to_A_infile_1,$C_to_T_infile_2,$G_to_A_infile_2);
|
|
378 }
|
|
379 ### Input files are in FastQ format
|
|
380 else{
|
|
381 process_fastQ_files_for_paired_end_methylation_calls($sequence_file_1,$sequence_file_2,$C_to_T_infile_1,$G_to_A_infile_1,$C_to_T_infile_2,$G_to_A_infile_2);
|
|
382 }
|
|
383 }
|
|
384
|
|
385 sub print_final_analysis_report_single_end{
|
|
386 my ($C_to_T_infile,$G_to_A_infile) = @_;
|
|
387 ### All sequences from the original sequence file have been analysed now
|
|
388 ### deleting temporary C->T or G->A infiles
|
|
389
|
|
390 if ($directional){
|
|
391 my $deletion_successful = unlink "$temp_dir$C_to_T_infile";
|
|
392 if ($deletion_successful == 1){
|
|
393 warn "\nSuccessfully deleted the temporary file $temp_dir$C_to_T_infile\n\n";
|
|
394 }
|
|
395 else{
|
|
396 warn "Could not delete temporary file $C_to_T_infile properly $!\n";
|
|
397 }
|
|
398 }
|
|
399
|
|
400 else{
|
|
401 my $deletion_successful = unlink "$temp_dir$C_to_T_infile","$temp_dir$G_to_A_infile";
|
|
402 if ($deletion_successful == 2){
|
|
403 warn "\nSuccessfully deleted the temporary files $temp_dir$C_to_T_infile and $temp_dir$G_to_A_infile\n\n";
|
|
404 }
|
|
405 else{
|
|
406 warn "Could not delete temporary files properly $!\n";
|
|
407 }
|
|
408 }
|
|
409
|
|
410 ### printing a final report for the alignment procedure
|
|
411 print REPORT "Final Alignment report\n",'='x22,"\n";
|
|
412 print "Final Alignment report\n",'='x22,"\n";
|
|
413 # foreach my $index (0..$#fhs){
|
|
414 # print "$fhs[$index]->{name}\n";
|
|
415 # print "$fhs[$index]->{seen}\talignments on the correct strand in total\n";
|
|
416 # print "$fhs[$index]->{wrong_strand}\talignments were discarded (nonsensical alignments)\n\n";
|
|
417 # }
|
|
418
|
|
419 ### printing a final report for the methylation call procedure
|
|
420 warn "Sequences analysed in total:\t$counting{sequences_count}\n";
|
|
421 print REPORT "Sequences analysed in total:\t$counting{sequences_count}\n";
|
|
422 my $percent_alignable_sequences;
|
|
423
|
|
424 if ($counting{sequences_count} == 0){
|
|
425 $percent_alignable_sequences = 0;
|
|
426 }
|
|
427 else{
|
|
428 $percent_alignable_sequences = sprintf ("%.1f",$counting{unique_best_alignment_count}*100/$counting{sequences_count});
|
|
429 }
|
|
430
|
|
431 warn "Number of alignments with a unique best hit from the different alignments:\t$counting{unique_best_alignment_count}\nMapping efficiency:\t${percent_alignable_sequences}%\n\n";
|
|
432 print REPORT "Number of alignments with a unique best hit from the different alignments:\t$counting{unique_best_alignment_count}\nMapping efficiency:\t${percent_alignable_sequences}%\n";
|
|
433
|
|
434 ### percentage of low complexity reads overruled because of low complexity (thereby creating a bias for highly methylated reads),
|
|
435 ### only calculating the percentage if there were any overruled alignments
|
|
436 if ($counting{low_complexity_alignments_overruled_count}){
|
|
437 my $percent_overruled_low_complexity_alignments = sprintf ("%.1f",$counting{low_complexity_alignments_overruled_count}*100/$counting{sequences_count});
|
|
438 # print REPORT "Number of low complexity alignments which were overruled to have a unique best hit rather than discarding them:\t$counting{low_complexity_alignments_overruled_count}\t(${percent_overruled_low_complexity_alignments}%)\n";
|
|
439 }
|
|
440
|
|
441 print "Sequences with no alignments under any condition:\t$counting{no_single_alignment_found}\n";
|
|
442 print "Sequences did not map uniquely:\t$counting{unsuitable_sequence_count}\n";
|
|
443 print "Sequences which were discarded because genomic sequence could not be extracted:\t$counting{genomic_sequence_could_not_be_extracted_count}\n\n";
|
|
444 print "Number of sequences with unique best (first) alignment came from the bowtie output:\n";
|
|
445 print join ("\n","CT/CT:\t$counting{CT_CT_count}\t((converted) top strand)","CT/GA:\t$counting{CT_GA_count}\t((converted) bottom strand)","GA/CT:\t$counting{GA_CT_count}\t(complementary to (converted) top strand)","GA/GA:\t$counting{GA_GA_count}\t(complementary to (converted) bottom strand)"),"\n\n";
|
|
446
|
|
447 print REPORT "Sequences with no alignments under any condition:\t$counting{no_single_alignment_found}\n";
|
|
448 print REPORT "Sequences did not map uniquely:\t$counting{unsuitable_sequence_count}\n";
|
|
449 print REPORT "Sequences which were discarded because genomic sequence could not be extracted:\t$counting{genomic_sequence_could_not_be_extracted_count}\n\n";
|
|
450 print REPORT "Number of sequences with unique best (first) alignment came from the bowtie output:\n";
|
|
451 print REPORT join ("\n","CT/CT:\t$counting{CT_CT_count}\t((converted) top strand)","CT/GA:\t$counting{CT_GA_count}\t((converted) bottom strand)","GA/CT:\t$counting{GA_CT_count}\t(complementary to (converted) top strand)","GA/GA:\t$counting{GA_GA_count}\t(complementary to (converted) bottom strand)"),"\n\n";
|
|
452
|
|
453 if ($directional){
|
|
454 print "Number of alignments to (merely theoretical) complementary strands being rejected in total:\t$counting{alignments_rejected_count}\n\n";
|
|
455 print REPORT "Number of alignments to (merely theoretical) complementary strands being rejected in total:\t$counting{alignments_rejected_count}\n\n";
|
|
456 }
|
|
457
|
|
458 ### detailed information about Cs analysed
|
|
459 warn "Final Cytosine Methylation Report\n",'='x33,"\n";
|
|
460 my $total_number_of_C = $counting{total_meCHH_count}+$counting{total_meCHG_count}+$counting{total_meCpG_count}+$counting{total_unmethylated_CHH_count}+$counting{total_unmethylated_CHG_count}+$counting{total_unmethylated_CpG_count};
|
|
461 warn "Total number of C's analysed:\t$total_number_of_C\n\n";
|
|
462 warn "Total methylated C's in CpG context:\t$counting{total_meCpG_count}\n";
|
|
463 warn "Total methylated C's in CHG context:\t$counting{total_meCHG_count}\n";
|
|
464 warn "Total methylated C's in CHH context:\t$counting{total_meCHH_count}\n\n";
|
|
465 warn "Total C to T conversions in CpG context:\t$counting{total_unmethylated_CpG_count}\n";
|
|
466 warn "Total C to T conversions in CHG context:\t$counting{total_unmethylated_CHG_count}\n";
|
|
467 warn "Total C to T conversions in CHH context:\t$counting{total_unmethylated_CHH_count}\n\n";
|
|
468
|
|
469 print REPORT "Final Cytosine Methylation Report\n",'='x33,"\n";
|
|
470 print REPORT "Total number of C's analysed:\t$total_number_of_C\n\n";
|
|
471 print REPORT "Total methylated C's in CpG context:\t $counting{total_meCpG_count}\n";
|
|
472 print REPORT "Total methylated C's in CHG context:\t$counting{total_meCHG_count}\n";
|
|
473 print REPORT "Total methylated C's in CHH context:\t$counting{total_meCHH_count}\n\n";
|
|
474 print REPORT "Total C to T conversions in CpG context:\t$counting{total_unmethylated_CpG_count}\n";
|
|
475 print REPORT "Total C to T conversions in CHG context:\t$counting{total_unmethylated_CHG_count}\n";
|
|
476 print REPORT "Total C to T conversions in CHH context:\t$counting{total_unmethylated_CHH_count}\n\n";
|
|
477
|
|
478 my $percent_meCHG;
|
|
479 if (($counting{total_meCHG_count}+$counting{total_unmethylated_CHG_count}) > 0){
|
|
480 $percent_meCHG = sprintf("%.1f",100*$counting{total_meCHG_count}/($counting{total_meCHG_count}+$counting{total_unmethylated_CHG_count}));
|
|
481 }
|
|
482
|
|
483 my $percent_meCHH;
|
|
484 if (($counting{total_meCHH_count}+$counting{total_unmethylated_CHH_count}) > 0){
|
|
485 $percent_meCHH = sprintf("%.1f",100*$counting{total_meCHH_count}/($counting{total_meCHH_count}+$counting{total_unmethylated_CHH_count}));
|
|
486 }
|
|
487
|
|
488 my $percent_meCpG;
|
|
489 if (($counting{total_meCpG_count}+$counting{total_unmethylated_CpG_count}) > 0){
|
|
490 $percent_meCpG = sprintf("%.1f",100*$counting{total_meCpG_count}/($counting{total_meCpG_count}+$counting{total_unmethylated_CpG_count}));
|
|
491 }
|
|
492
|
|
493 ### printing methylated CpG percentage if applicable
|
|
494 if ($percent_meCpG){
|
|
495 warn "C methylated in CpG context:\t${percent_meCpG}%\n";
|
|
496 print REPORT "C methylated in CpG context:\t${percent_meCpG}%\n";
|
|
497 }
|
|
498 else{
|
|
499 warn "Can't determine percentage of methylated Cs in CpG context if value was 0\n";
|
|
500 print REPORT "Can't determine percentage of methylated Cs in CpG context if value was 0\n";
|
|
501 }
|
|
502
|
|
503 ### printing methylated C percentage (CHG context) if applicable
|
|
504 if ($percent_meCHG){
|
|
505 warn "C methylated in CHG context:\t${percent_meCHG}%\n";
|
|
506 print REPORT "C methylated in CHG context:\t${percent_meCHG}%\n";
|
|
507 }
|
|
508 else{
|
|
509 warn "Can't determine percentage of methylated Cs in CHG context if value was 0\n";
|
|
510 print REPORT "Can't determine percentage of methylated Cs in CHG context if value was 0\n";
|
|
511 }
|
|
512
|
|
513 ### printing methylated C percentage (CHH context) if applicable
|
|
514 if ($percent_meCHH){
|
|
515 warn "C methylated in CHH context:\t${percent_meCHH}%\n\n\n";
|
|
516 print REPORT "C methylated in CHH context:\t${percent_meCHH}%\n\n\n";
|
|
517 }
|
|
518 else{
|
|
519 warn "Can't determine percentage of methylated Cs in CHH context if value was 0\n\n\n";
|
|
520 print REPORT "Can't determine percentage of methylated Cs in CHH context if value was 0\n\n\n";
|
|
521 }
|
|
522
|
|
523 if ($seqID_contains_tabs){
|
|
524 warn "The sequence IDs in the provided file contain tab-stops which might prevent sequence alignments. If this happened, please replace all tab characters within the seqID field with spaces before running Bismark.\n\n";
|
|
525 print REPORT "The sequence IDs in the provided file contain tab-stops which might prevent sequence alignments. If this happened, please replace all tab characters within the seqID field with spaces before running Bismark.\n\n";
|
|
526 }
|
|
527 }
|
|
528
|
|
529 sub print_final_analysis_report_paired_ends{
|
|
530 my ($C_to_T_infile_1,$G_to_A_infile_1,$C_to_T_infile_2,$G_to_A_infile_2) = @_;
|
|
531 ### All sequences from the original sequence file have been analysed now, therefore deleting temporary C->T or G->A infiles
|
|
532 if ($directional){
|
|
533 my $deletion_successful = unlink "$temp_dir$C_to_T_infile_1","$temp_dir$G_to_A_infile_2";
|
|
534 if ($deletion_successful == 2){
|
|
535 warn "\nSuccessfully deleted the temporary files $temp_dir$C_to_T_infile_1 and $temp_dir$G_to_A_infile_2\n\n";
|
|
536 }
|
|
537 else{
|
|
538 warn "Could not delete temporary files $temp_dir$C_to_T_infile_1 and $temp_dir$G_to_A_infile_2 properly: $!\n";
|
|
539 }
|
|
540 }
|
|
541 else{
|
|
542 my $deletion_successful = unlink "$temp_dir$C_to_T_infile_1","$temp_dir$G_to_A_infile_1","$temp_dir$C_to_T_infile_2","$temp_dir$G_to_A_infile_2";
|
|
543 if ($deletion_successful == 4){
|
|
544 warn "\nSuccessfully deleted the temporary files $temp_dir$C_to_T_infile_1, $temp_dir$G_to_A_infile_1, $temp_dir$C_to_T_infile_2 and $temp_dir$G_to_A_infile_2\n\n";
|
|
545 }
|
|
546 else{
|
|
547 warn "Could not delete temporary files properly: $!\n";
|
|
548 }
|
|
549 }
|
|
550
|
|
551 ### printing a final report for the alignment procedure
|
|
552 warn "Final Alignment report\n",'='x22,"\n";
|
|
553 print REPORT "Final Alignment report\n",'='x22,"\n";
|
|
554 # foreach my $index (0..$#fhs){
|
|
555 # print "$fhs[$index]->{name}\n";
|
|
556 # print "$fhs[$index]->{seen}\talignments on the correct strand in total\n";
|
|
557 # print "$fhs[$index]->{wrong_strand}\talignments were discarded (nonsensical alignments)\n\n";
|
|
558 # }
|
|
559
|
|
560 ### printing a final report for the methylation call procedure
|
|
561 warn "Sequence pairs analysed in total:\t$counting{sequences_count}\n";
|
|
562 print REPORT "Sequence pairs analysed in total:\t$counting{sequences_count}\n";
|
|
563
|
|
564 my $percent_alignable_sequence_pairs;
|
|
565 if ($counting{sequences_count} == 0){
|
|
566 $percent_alignable_sequence_pairs = 0;
|
|
567 }
|
|
568 else{
|
|
569 $percent_alignable_sequence_pairs = sprintf ("%.1f",$counting{unique_best_alignment_count}*100/$counting{sequences_count});
|
|
570 }
|
|
571 print "Number of paired-end alignments with a unique best hit:\t$counting{unique_best_alignment_count}\nMapping efficiency:\t${percent_alignable_sequence_pairs}%\n\n";
|
|
572 print REPORT "Number of paired-end alignments with a unique best hit:\t$counting{unique_best_alignment_count}\nMapping efficiency:\t${percent_alignable_sequence_pairs}% \n";
|
|
573
|
|
574 print "Sequence pairs with no alignments under any condition:\t$counting{no_single_alignment_found}\n";
|
|
575 print "Sequence pairs did not map uniquely:\t$counting{unsuitable_sequence_count}\n";
|
|
576 print "Sequence pairs which were discarded because genomic sequence could not be extracted:\t$counting{genomic_sequence_could_not_be_extracted_count}\n\n";
|
|
577 print "Number of sequence pairs with unique best (first) alignment came from the bowtie output:\n";
|
|
578 print join ("\n","CT/GA/CT:\t$counting{CT_GA_CT_count}\t((converted) top strand)","GA/CT/CT:\t$counting{GA_CT_CT_count}\t(complementary to (converted) top strand)","GA/CT/GA:\t$counting{GA_CT_GA_count}\t(complementary to (converted) bottom strand)","CT/GA/GA:\t$counting{CT_GA_GA_count}\t((converted) bottom strand)"),"\n\n";
|
|
579
|
|
580
|
|
581 print REPORT "Sequence pairs with no alignments under any condition:\t$counting{no_single_alignment_found}\n";
|
|
582 print REPORT "Sequence pairs did not map uniquely:\t$counting{unsuitable_sequence_count}\n";
|
|
583 print REPORT "Sequence pairs which were discarded because genomic sequence could not be extracted:\t$counting{genomic_sequence_could_not_be_extracted_count}\n\n";
|
|
584 print REPORT "Number of sequence pairs with unique best (first) alignment came from the bowtie output:\n";
|
|
585 print REPORT join ("\n","CT/GA/CT:\t$counting{CT_GA_CT_count}\t((converted) top strand)","GA/CT/CT:\t$counting{GA_CT_CT_count}\t(complementary to (converted) top strand)","GA/CT/GA:\t$counting{GA_CT_GA_count}\t(complementary to (converted) bottom strand)","CT/GA/GA:\t$counting{CT_GA_GA_count}\t((converted) bottom strand)"),"\n\n";
|
|
586 ### detailed information about Cs analysed
|
|
587
|
|
588 if ($directional){
|
|
589 print "Number of alignments to (merely theoretical) complementary strands being rejected in total:\t$counting{alignments_rejected_count}\n\n";
|
|
590 print REPORT "Number of alignments to (merely theoretical) complementary strands being rejected in total:\t$counting{alignments_rejected_count}\n\n";
|
|
591 }
|
|
592
|
|
593 warn "Final Cytosine Methylation Report\n",'='x33,"\n";
|
|
594 print REPORT "Final Cytosine Methylation Report\n",'='x33,"\n";
|
|
595
|
|
596 my $total_number_of_C = $counting{total_meCHG_count}+ $counting{total_meCHH_count}+$counting{total_meCpG_count}+$counting{total_unmethylated_CHG_count}+$counting{total_unmethylated_CHH_count}+$counting{total_unmethylated_CpG_count};
|
|
597 warn "Total number of C's analysed:\t$total_number_of_C\n\n";
|
|
598 warn "Total methylated C's in CpG context:\t$counting{total_meCpG_count}\n";
|
|
599 warn "Total methylated C's in CHG context:\t$counting{total_meCHG_count}\n";
|
|
600 warn "Total methylated C's in CHH context:\t$counting{total_meCHH_count}\n\n";
|
|
601 warn "Total C to T conversions in CpG context:\t$counting{total_unmethylated_CpG_count}\n";
|
|
602 warn "Total C to T conversions in CHG context:\t$counting{total_unmethylated_CHG_count}\n";
|
|
603 warn "Total C to T conversions in CHH context:\t$counting{total_unmethylated_CHH_count}\n\n";
|
|
604
|
|
605 print REPORT "Total number of C's analysed:\t$total_number_of_C\n\n";
|
|
606 print REPORT "Total methylated C's in CpG context:\t$counting{total_meCpG_count}\n";
|
|
607 print REPORT "Total methylated C's in CHG context:\t$counting{total_meCHG_count}\n";
|
|
608 print REPORT "Total methylated C's in CHH context:\t$counting{total_meCHH_count}\n\n";
|
|
609 print REPORT "Total C to T conversions in CpG context:\t$counting{total_unmethylated_CpG_count}\n";
|
|
610 print REPORT "Total C to T conversions in CHG context:\t$counting{total_unmethylated_CHG_count}\n";
|
|
611 print REPORT "Total C to T conversions in CHH context:\t$counting{total_unmethylated_CHH_count}\n\n";
|
|
612
|
|
613 my $percent_meCHG;
|
|
614 if (($counting{total_meCHG_count}+$counting{total_unmethylated_CHG_count}) > 0){
|
|
615 $percent_meCHG = sprintf("%.1f",100*$counting{total_meCHG_count}/($counting{total_meCHG_count}+$counting{total_unmethylated_CHG_count}));
|
|
616 }
|
|
617
|
|
618 my $percent_meCHH;
|
|
619 if (($counting{total_meCHH_count}+$counting{total_unmethylated_CHH_count}) > 0){
|
|
620 $percent_meCHH = sprintf("%.1f",100*$counting{total_meCHH_count}/($counting{total_meCHH_count}+$counting{total_unmethylated_CHH_count}));
|
|
621 }
|
|
622
|
|
623 my $percent_meCpG;
|
|
624 if (($counting{total_meCpG_count}+$counting{total_unmethylated_CpG_count}) > 0){
|
|
625 $percent_meCpG = sprintf("%.1f",100*$counting{total_meCpG_count}/($counting{total_meCpG_count}+$counting{total_unmethylated_CpG_count}));
|
|
626 }
|
|
627
|
|
628 ### printing methylated CpG percentage if applicable
|
|
629 if ($percent_meCpG){
|
|
630 warn "C methylated in CpG context:\t${percent_meCpG}%\n";
|
|
631 print REPORT "C methylated in CpG context:\t${percent_meCpG}%\n";
|
|
632 }
|
|
633 else{
|
|
634 warn "Can't determine percentage of methylated Cs in CpG context if value was 0\n";
|
|
635 print REPORT "Can't determine percentage of methylated Cs in CpG context if value was 0\n";
|
|
636 }
|
|
637
|
|
638 ### printing methylated C percentage in CHG context if applicable
|
|
639 if ($percent_meCHG){
|
|
640 warn "C methylated in CHG context:\t${percent_meCHG}%\n";
|
|
641 print REPORT "C methylated in CHG context:\t${percent_meCHG}%\n";
|
|
642 }
|
|
643 else{
|
|
644 warn "Can't determine percentage of methylated Cs in CHG context if value was 0\n";
|
|
645 print REPORT "Can't determine percentage of methylated Cs in CHG context if value was 0\n";
|
|
646 }
|
|
647
|
|
648 ### printing methylated C percentage in CHH context if applicable
|
|
649 if ($percent_meCHH){
|
|
650 warn "C methylated in CHH context:\t${percent_meCHH}%\n\n\n";
|
|
651 print REPORT "C methylated in CHH context:\t${percent_meCHH}%\n\n\n";
|
|
652 }
|
|
653 else{
|
|
654 warn "Can't determine percentage of methylated Cs in CHH context if value was 0\n\n\n";
|
|
655 print REPORT "Can't determine percentage of methylated Cs in CHH context if value was 0\n\n\n";
|
|
656 }
|
|
657
|
|
658 }
|
|
659
|
|
660 sub process_single_end_fastA_file_for_methylation_call{
|
|
661 my ($sequence_file,$C_to_T_infile,$G_to_A_infile) = @_;
|
|
662 ### this is a FastA sequence file; we need the actual sequence to compare it against the genomic sequence in order to make a methylation call.
|
|
663 ### Now reading in the sequence file sequence by sequence and see if the current sequence was mapped to one (or both) of the converted genomes in either
|
|
664 ### the C->T or G->A version
|
|
665
|
|
666 ### gzipped version of the infile
|
|
667 if ($sequence_file =~ /\.gz$/){
|
|
668 open (IN,"zcat $sequence_file |") or die $!;
|
|
669 }
|
|
670 else{
|
|
671 open (IN,$sequence_file) or die $!;
|
|
672 }
|
|
673
|
|
674 my $count = 0;
|
|
675
|
|
676 warn "\nReading in the sequence file $sequence_file\n";
|
|
677 while (1) {
|
|
678 # last if ($counting{sequences_count} > 100);
|
|
679 my $identifier = <IN>;
|
|
680 my $sequence = <IN>;
|
|
681 last unless ($identifier and $sequence);
|
|
682
|
|
683 $identifier = fix_IDs($identifier); # this is to avoid problems with truncated read ID when they contain white spaces
|
|
684
|
|
685 ++$count;
|
|
686
|
|
687 if ($skip){
|
|
688 next unless ($count > $skip);
|
|
689 }
|
|
690 if ($upto){
|
|
691 last if ($count > $upto);
|
|
692 }
|
|
693
|
|
694 $counting{sequences_count}++;
|
|
695 if ($counting{sequences_count}%100000==0) {
|
|
696 warn "Processed $counting{sequences_count} sequences so far\n";
|
|
697 }
|
|
698 chomp $sequence;
|
|
699 chomp $identifier;
|
|
700
|
|
701 $identifier =~ s/^>//; # deletes the > at the beginning of FastA headers
|
|
702
|
|
703 my $return;
|
|
704 if ($bowtie2){
|
|
705 $return = check_bowtie_results_single_end_bowtie2 (uc$sequence,$identifier);
|
|
706 }
|
|
707 else{
|
|
708 $return = check_bowtie_results_single_end(uc$sequence,$identifier); # default Bowtie 1
|
|
709 }
|
|
710
|
|
711 unless ($return){
|
|
712 $return = 0;
|
|
713 }
|
|
714
|
|
715 # print the sequence to ambiguous.out if --ambiguous was specified
|
|
716 if ($ambiguous and $return == 2){
|
|
717 print AMBIG ">$identifier\n";
|
|
718 print AMBIG "$sequence\n";
|
|
719 }
|
|
720
|
|
721 # print the sequence to <unmapped.out> file if --un was specified
|
|
722 elsif ($unmapped and $return == 1){
|
|
723 print UNMAPPED ">$identifier\n";
|
|
724 print UNMAPPED "$sequence\n";
|
|
725 }
|
|
726 }
|
|
727 print "Processed $counting{sequences_count} sequences in total\n\n";
|
|
728
|
|
729 print_final_analysis_report_single_end($C_to_T_infile,$G_to_A_infile);
|
|
730
|
|
731 }
|
|
732
|
|
733 sub process_single_end_fastQ_file_for_methylation_call{
|
|
734 my ($sequence_file,$C_to_T_infile,$G_to_A_infile) = @_;
|
|
735 ### this is the Illumina sequence file; we need the actual sequence to compare it against the genomic sequence in order to make a methylation call.
|
|
736 ### Now reading in the sequence file sequence by sequence and see if the current sequence was mapped to one (or both) of the converted genomes in either
|
|
737 ### the C->T or G->A version
|
|
738
|
|
739 ### gzipped version of the infile
|
|
740 if ($sequence_file =~ /\.gz$/){
|
|
741 open (IN,"zcat $sequence_file |") or die $!;
|
|
742 }
|
|
743 else{
|
|
744 open (IN,$sequence_file) or die $!;
|
|
745 }
|
|
746
|
|
747 my $count = 0;
|
|
748
|
|
749 warn "\nReading in the sequence file $sequence_file\n";
|
|
750 while (1) {
|
|
751 my $identifier = <IN>;
|
|
752 my $sequence = <IN>;
|
|
753 my $identifier_2 = <IN>;
|
|
754 my $quality_value = <IN>;
|
|
755 last unless ($identifier and $sequence and $identifier_2 and $quality_value);
|
|
756
|
|
757 $identifier = fix_IDs($identifier); # this is to avoid problems with truncated read ID when they contain white spaces
|
|
758
|
|
759 ++$count;
|
|
760
|
|
761 if ($skip){
|
|
762 next unless ($count > $skip);
|
|
763 }
|
|
764 if ($upto){
|
|
765 last if ($count > $upto);
|
|
766 }
|
|
767
|
|
768 $counting{sequences_count}++;
|
|
769
|
|
770 if ($counting{sequences_count}%1000000==0) {
|
|
771 warn "Processed $counting{sequences_count} sequences so far\n";
|
|
772 }
|
|
773 chomp $sequence;
|
|
774 chomp $identifier;
|
|
775 chomp $quality_value;
|
|
776
|
|
777 $identifier =~ s/^\@//; # deletes the @ at the beginning of Illumin FastQ headers
|
|
778
|
|
779 my $return;
|
|
780 if ($bowtie2){
|
|
781 $return = check_bowtie_results_single_end_bowtie2 (uc$sequence,$identifier,$quality_value);
|
|
782 }
|
|
783 else{
|
|
784 $return = check_bowtie_results_single_end(uc$sequence,$identifier,$quality_value); # default Bowtie 1
|
|
785 }
|
|
786
|
|
787 unless ($return){
|
|
788 $return = 0;
|
|
789 }
|
|
790
|
|
791 # print the sequence to ambiguous.out if --ambiguous was specified
|
|
792 if ($ambiguous and $return == 2){
|
|
793 print AMBIG "\@$identifier\n";
|
|
794 print AMBIG "$sequence\n";
|
|
795 print AMBIG $identifier_2;
|
|
796 print AMBIG "$quality_value\n";
|
|
797 }
|
|
798
|
|
799 # print the sequence to <unmapped.out> file if --un was specified
|
|
800 elsif ($unmapped and $return == 1){
|
|
801 print UNMAPPED "\@$identifier\n";
|
|
802 print UNMAPPED "$sequence\n";
|
|
803 print UNMAPPED $identifier_2;
|
|
804 print UNMAPPED "$quality_value\n";
|
|
805 }
|
|
806 }
|
|
807 print "Processed $counting{sequences_count} sequences in total\n\n";
|
|
808
|
|
809 print_final_analysis_report_single_end($C_to_T_infile,$G_to_A_infile);
|
|
810
|
|
811 }
|
|
812
|
|
813 sub process_fastA_files_for_paired_end_methylation_calls{
|
|
814 my ($sequence_file_1,$sequence_file_2,$C_to_T_infile_1,$G_to_A_infile_1,$C_to_T_infile_2,$G_to_A_infile_2) = @_;
|
|
815 ### Processing the two FastA sequence files; we need the actual sequences of both reads to compare them against the genomic sequence in order to
|
|
816 ### make a methylation call. The sequence idetifier per definition needs to be the same for a sequence pair used for paired-end mapping.
|
|
817 ### Now reading in the sequence files sequence by sequence and see if the current sequences produced an alignment to one (or both) of the
|
|
818 ### converted genomes (either the C->T or G->A version)
|
|
819
|
|
820 ### gzipped version of the infiles
|
|
821 if ($sequence_file_1 =~ /\.gz$/ and $sequence_file_2 =~ /\.gz$/){
|
|
822 open (IN1,"zcat $sequence_file_1 |") or die "Failed to open zcat pipe to $sequence_file_1 $!\n";
|
|
823 open (IN2,"zcat $sequence_file_2 |") or die "Failed to open zcat pipe to $sequence_file_2 $!\n";
|
|
824 }
|
|
825 else{
|
|
826 open (IN1,$sequence_file_1) or die $!;
|
|
827 open (IN2,$sequence_file_2) or die $!;
|
|
828 }
|
|
829
|
|
830 warn "\nReading in the sequence files $sequence_file_1 and $sequence_file_2\n";
|
|
831 ### Both files are required to have the exact same number of sequences, therefore we can process the sequences jointly one by one
|
|
832
|
|
833 my $count = 0;
|
|
834
|
|
835 while (1) {
|
|
836 # reading from the first input file
|
|
837 my $identifier_1 = <IN1>;
|
|
838 my $sequence_1 = <IN1>;
|
|
839 # reading from the second input file
|
|
840 my $identifier_2 = <IN2>;
|
|
841 my $sequence_2 = <IN2>;
|
|
842 last unless ($identifier_1 and $sequence_1 and $identifier_2 and $sequence_2);
|
|
843
|
|
844 $identifier_1 = fix_IDs($identifier_1); # this is to avoid problems with truncated read ID when they contain white spaces
|
|
845 $identifier_2 = fix_IDs($identifier_2);
|
|
846
|
|
847 ++$count;
|
|
848
|
|
849 if ($skip){
|
|
850 next unless ($count > $skip);
|
|
851 }
|
|
852 if ($upto){
|
|
853 last if ($count > $upto);
|
|
854 }
|
|
855
|
|
856 $counting{sequences_count}++;
|
|
857 if ($counting{sequences_count}%100000==0) {
|
|
858 warn "Processed $counting{sequences_count} sequences so far\n";
|
|
859 }
|
|
860 my $orig_identifier_1 = $identifier_1;
|
|
861 my $orig_identifier_2 = $identifier_2;
|
|
862
|
|
863 chomp $sequence_1;
|
|
864 chomp $identifier_1;
|
|
865 chomp $sequence_2;
|
|
866 chomp $identifier_2;
|
|
867
|
|
868 $identifier_1 =~ s/^>//; # deletes the > at the beginning of FastA headers
|
|
869
|
|
870 my $return;
|
|
871 if ($bowtie2){
|
|
872 $return = check_bowtie_results_paired_ends_bowtie2 (uc$sequence_1,uc$sequence_2,$identifier_1);
|
|
873 }
|
|
874 else{
|
|
875 $return = check_bowtie_results_paired_ends (uc$sequence_1,uc$sequence_2,$identifier_1);
|
|
876 }
|
|
877
|
|
878 unless ($return){
|
|
879 $return = 0;
|
|
880 }
|
|
881
|
|
882 # print the sequences to ambiguous_1 and _2 if --ambiguous was specified
|
|
883 if ($ambiguous and $return == 2){
|
|
884 print AMBIG_1 $orig_identifier_1;
|
|
885 print AMBIG_1 "$sequence_1\n";
|
|
886 print AMBIG_2 $orig_identifier_2;
|
|
887 print AMBIG_2 "$sequence_2\n";
|
|
888 }
|
|
889
|
|
890 # print the sequences to unmapped_1.out and unmapped_2.out if --un was specified
|
|
891 elsif ($unmapped and $return == 1){
|
|
892 print UNMAPPED_1 $orig_identifier_1;
|
|
893 print UNMAPPED_1 "$sequence_1\n";
|
|
894 print UNMAPPED_2 $orig_identifier_2;
|
|
895 print UNMAPPED_2 "$sequence_2\n";
|
|
896 }
|
|
897 }
|
|
898
|
|
899 print "Processed $counting{sequences_count} sequences in total\n\n";
|
|
900
|
|
901 print_final_analysis_report_paired_ends($C_to_T_infile_1,$G_to_A_infile_1,$C_to_T_infile_2,$G_to_A_infile_2);
|
|
902
|
|
903 }
|
|
904
|
|
905 sub process_fastQ_files_for_paired_end_methylation_calls{
|
|
906 my ($sequence_file_1,$sequence_file_2,$C_to_T_infile_1,$G_to_A_infile_1,$C_to_T_infile_2,$G_to_A_infile_2) = @_;
|
|
907 ### Processing the two Illumina sequence files; we need the actual sequence of both reads to compare them against the genomic sequence in order to
|
|
908 ### make a methylation call. The sequence identifier per definition needs to be same for a sequence pair used for paired-end alignments.
|
|
909 ### Now reading in the sequence files sequence by sequence and see if the current sequences produced a paired-end alignment to one (or both)
|
|
910 ### of the converted genomes (either C->T or G->A version)
|
|
911
|
|
912 ### gzipped version of the infiles
|
|
913 if ($sequence_file_1 =~ /\.gz$/ and $sequence_file_2 =~ /\.gz$/){
|
|
914 open (IN1,"zcat $sequence_file_1 |") or die "Failed to open zcat pipe to $sequence_file_1 $!\n";
|
|
915 open (IN2,"zcat $sequence_file_2 |") or die "Failed to open zcat pipe to $sequence_file_2 $!\n";
|
|
916 }
|
|
917 else{
|
|
918 open (IN1,$sequence_file_1) or die $!;
|
|
919 open (IN2,$sequence_file_2) or die $!;
|
|
920 }
|
|
921
|
|
922 my $count = 0;
|
|
923
|
|
924 warn "\nReading in the sequence files $sequence_file_1 and $sequence_file_2\n";
|
|
925 ### Both files are required to have the exact same number of sequences, therefore we can process the sequences jointly one by one
|
|
926 while (1) {
|
|
927 # reading from the first input file
|
|
928 my $identifier_1 = <IN1>;
|
|
929 my $sequence_1 = <IN1>;
|
|
930 my $ident_1 = <IN1>; # not needed
|
|
931 my $quality_value_1 = <IN1>; # not needed
|
|
932 # reading from the second input file
|
|
933 my $identifier_2 = <IN2>;
|
|
934 my $sequence_2 = <IN2>;
|
|
935 my $ident_2 = <IN2>; # not needed
|
|
936 my $quality_value_2 = <IN2>; # not needed
|
|
937 last unless ($identifier_1 and $sequence_1 and $quality_value_1 and $identifier_2 and $sequence_2 and $quality_value_2);
|
|
938
|
|
939 $identifier_1 = fix_IDs($identifier_1); # this is to avoid problems with truncated read ID when they contain white spaces
|
|
940 $identifier_2 = fix_IDs($identifier_2);
|
|
941
|
|
942 ++$count;
|
|
943
|
|
944 if ($skip){
|
|
945 next unless ($count > $skip);
|
|
946 }
|
|
947 if ($upto){
|
|
948 last if ($count > $upto);
|
|
949 }
|
|
950
|
|
951 $counting{sequences_count}++;
|
|
952 if ($counting{sequences_count}%100000==0) {
|
|
953 warn "Processed $counting{sequences_count} sequences so far\n";
|
|
954 }
|
|
955
|
|
956 my $orig_identifier_1 = $identifier_1;
|
|
957 my $orig_identifier_2 = $identifier_2;
|
|
958
|
|
959 chomp $sequence_1;
|
|
960 chomp $identifier_1;
|
|
961 chomp $sequence_2;
|
|
962 chomp $identifier_2;
|
|
963 chomp $quality_value_1;
|
|
964 chomp $quality_value_2;
|
|
965
|
|
966 $identifier_1 =~ s/^\@//; # deletes the @ at the beginning of the FastQ ID
|
|
967
|
|
968 my $return;
|
|
969 if ($bowtie2){
|
|
970 $return = check_bowtie_results_paired_ends_bowtie2 (uc$sequence_1,uc$sequence_2,$identifier_1,$quality_value_1,$quality_value_2);
|
|
971 }
|
|
972 else{
|
|
973 $return = check_bowtie_results_paired_ends (uc$sequence_1,uc$sequence_2,$identifier_1,$quality_value_1,$quality_value_2);
|
|
974 }
|
|
975
|
|
976 unless ($return){
|
|
977 $return = 0;
|
|
978 }
|
|
979
|
|
980 # print the sequences to ambiguous_1 and _2 if --ambiguous was specified
|
|
981 if ($ambiguous and $return == 2){
|
|
982 # seq_1
|
|
983 print AMBIG_1 $orig_identifier_1;
|
|
984 print AMBIG_1 "$sequence_1\n";
|
|
985 print AMBIG_1 $ident_1;
|
|
986 print AMBIG_1 "$quality_value_1\n";
|
|
987 # seq_2
|
|
988 print AMBIG_2 $orig_identifier_2;
|
|
989 print AMBIG_2 "$sequence_2\n";
|
|
990 print AMBIG_2 $ident_2;
|
|
991 print AMBIG_2 "$quality_value_2\n";
|
|
992 }
|
|
993
|
|
994 # print the sequences to unmapped_1.out and unmapped_2.out if --un was specified
|
|
995 elsif ($unmapped and $return == 1){
|
|
996 # seq_1
|
|
997 print UNMAPPED_1 $orig_identifier_1;
|
|
998 print UNMAPPED_1 "$sequence_1\n";
|
|
999 print UNMAPPED_1 $ident_1;
|
|
1000 print UNMAPPED_1 "$quality_value_1\n";
|
|
1001 # seq_2
|
|
1002 print UNMAPPED_2 $orig_identifier_2;
|
|
1003 print UNMAPPED_2 "$sequence_2\n";
|
|
1004 print UNMAPPED_2 $ident_2;
|
|
1005 print UNMAPPED_2 "$quality_value_2\n";
|
|
1006 }
|
|
1007 }
|
|
1008
|
|
1009 print "Processed $counting{sequences_count} sequences in total\n\n";
|
|
1010
|
|
1011 print_final_analysis_report_paired_ends($C_to_T_infile_1,$G_to_A_infile_1,$C_to_T_infile_2,$G_to_A_infile_2);
|
|
1012
|
|
1013 }
|
|
1014
|
|
1015 sub check_bowtie_results_single_end{
|
|
1016 my ($sequence,$identifier,$quality_value) = @_;
|
|
1017
|
|
1018 unless ($quality_value){ # FastA sequences get assigned a quality value of Phred 40 throughout
|
|
1019 $quality_value = 'I'x(length$sequence);
|
|
1020 }
|
|
1021
|
|
1022 my %mismatches = ();
|
|
1023 ### reading from the bowtie output files to see if this sequence aligned to a bisulfite converted genome
|
|
1024 foreach my $index (0..$#fhs){
|
|
1025
|
|
1026 ### skipping this index if the last alignment has been set to undefined already (i.e. end of bowtie output)
|
|
1027 next unless ($fhs[$index]->{last_line} and defined $fhs[$index]->{last_seq_id});
|
|
1028 ### if the sequence we are currently looking at produced an alignment we are doing various things with it
|
|
1029 if ($fhs[$index]->{last_seq_id} eq $identifier) {
|
|
1030 ###############################################################
|
|
1031 ### STEP I Now processing the alignment stored in last_line ###
|
|
1032 ###############################################################
|
|
1033 my $valid_alignment_found_1 = decide_whether_single_end_alignment_is_valid($index,$identifier);
|
|
1034 ### sequences can fail at this point if there was only 1 seq in the wrong orientation, or if there were 2 seqs, both in the wrong orientation
|
|
1035 ### we only continue to extract useful information about this alignment if 1 was returned
|
|
1036 if ($valid_alignment_found_1 == 1){
|
|
1037 ### Bowtie outputs which made it this far are in the correct orientation, so we can continue to analyse the alignment itself
|
|
1038 ### need to extract the chromosome number from the bowtie output (which is either XY_cf (complete forward) or XY_cr (complete reverse)
|
|
1039 my ($id,$strand,$mapped_chromosome,$position,$bowtie_sequence,$mismatch_info) = (split (/\t/,$fhs[$index]->{last_line},-1))[0,1,2,3,4,7];
|
|
1040
|
|
1041 unless($mismatch_info){
|
|
1042 $mismatch_info = '';
|
|
1043 }
|
|
1044
|
|
1045 chomp $mismatch_info;
|
|
1046 my $chromosome;
|
|
1047 if ($mapped_chromosome =~ s/_(CT|GA)_converted$//){
|
|
1048 $chromosome = $mapped_chromosome;
|
|
1049 }
|
|
1050 else{
|
|
1051 die "Chromosome number extraction failed for $mapped_chromosome\n";
|
|
1052 }
|
|
1053 ### Now extracting the number of mismatches to the converted genome
|
|
1054 my $number_of_mismatches;
|
|
1055 if ($mismatch_info eq ''){
|
|
1056 $number_of_mismatches = 0;
|
|
1057 }
|
|
1058 elsif ($mismatch_info =~ /^\d/){
|
|
1059 my @mismatches = split (/,/,$mismatch_info);
|
|
1060 $number_of_mismatches = scalar @mismatches;
|
|
1061 }
|
|
1062 else{
|
|
1063 die "Something weird is going on with the mismatch field:\t>>> $mismatch_info <<<\n";
|
|
1064 }
|
|
1065 ### creating a composite location variable from $chromosome and $position and storing the alignment information in a temporary hash table
|
|
1066 my $alignment_location = join (":",$chromosome,$position);
|
|
1067 ### If a sequence aligns to exactly the same location twice the sequence does either not contain any C or G, or all the Cs (or Gs on the reverse
|
|
1068 ### strand) were methylated and therefore protected. It is not needed to overwrite the same positional entry with a second entry for the same
|
|
1069 ### location (the genomic sequence extraction and methylation would not be affected by this, only the thing which would change is the index
|
|
1070 ### number for the found alignment)
|
|
1071 unless (exists $mismatches{$number_of_mismatches}->{$alignment_location}){
|
|
1072 $mismatches{$number_of_mismatches}->{$alignment_location}->{seq_id}=$id;
|
|
1073 $mismatches{$number_of_mismatches}->{$alignment_location}->{bowtie_sequence}=$bowtie_sequence;
|
|
1074 $mismatches{$number_of_mismatches}->{$alignment_location}->{index}=$index;
|
|
1075 $mismatches{$number_of_mismatches}->{$alignment_location}->{chromosome}=$chromosome;
|
|
1076 $mismatches{$number_of_mismatches}->{$alignment_location}->{position}=$position;
|
|
1077 }
|
|
1078 $number_of_mismatches = undef;
|
|
1079 ##################################################################################################################################################
|
|
1080 ### STEP II Now reading in the next line from the bowtie filehandle. The next alignment can either be a second alignment of the same sequence or a
|
|
1081 ### a new sequence. In either case we will store the next line in @fhs ->{last_line}. In case the alignment is already the next entry, a 0 will
|
|
1082 ### be returned as $valid_alignment_found and it will then be processed in the next round only.
|
|
1083 ##################################################################################################################################################
|
|
1084 my $newline = $fhs[$index]->{fh}-> getline();
|
|
1085 if ($newline){
|
|
1086 my ($seq_id) = split (/\t/,$newline);
|
|
1087 $fhs[$index]->{last_seq_id} = $seq_id;
|
|
1088 $fhs[$index]->{last_line} = $newline;
|
|
1089 }
|
|
1090 else {
|
|
1091 # assigning undef to last_seq_id and last_line and jumping to the next index (end of bowtie output)
|
|
1092 $fhs[$index]->{last_seq_id} = undef;
|
|
1093 $fhs[$index]->{last_line} = undef;
|
|
1094 next;
|
|
1095 }
|
|
1096 my $valid_alignment_found_2 = decide_whether_single_end_alignment_is_valid($index,$identifier);
|
|
1097 ### we only continue to extract useful information about this second alignment if 1 was returned
|
|
1098 if ($valid_alignment_found_2 == 1){
|
|
1099 ### If the second Bowtie output made it this far it is in the correct orientation, so we can continue to analyse the alignment itself
|
|
1100 ### need to extract the chromosome number from the bowtie output (which is either XY_cf (complete forward) or XY_cr (complete reverse)
|
|
1101 my ($id,$strand,$mapped_chromosome,$position,$bowtie_sequence,$mismatch_info) = (split (/\t/,$fhs[$index]->{last_line},-1))[0,1,2,3,4,7];
|
|
1102 unless($mismatch_info){
|
|
1103 $mismatch_info = '';
|
|
1104 }
|
|
1105 chomp $mismatch_info;
|
|
1106
|
|
1107 my $chromosome;
|
|
1108 if ($mapped_chromosome =~ s/_(CT|GA)_converted$//){
|
|
1109 $chromosome = $mapped_chromosome;
|
|
1110 }
|
|
1111 else{
|
|
1112 die "Chromosome number extraction failed for $mapped_chromosome\n";
|
|
1113 }
|
|
1114
|
|
1115 ### Now extracting the number of mismatches to the converted genome
|
|
1116 my $number_of_mismatches;
|
|
1117 if ($mismatch_info eq ''){
|
|
1118 $number_of_mismatches = 0;
|
|
1119 }
|
|
1120 elsif ($mismatch_info =~ /^\d/){
|
|
1121 my @mismatches = split (/,/,$mismatch_info);
|
|
1122 $number_of_mismatches = scalar @mismatches;
|
|
1123 }
|
|
1124 else{
|
|
1125 die "Something weird is going on with the mismatch field\n";
|
|
1126 }
|
|
1127 ### creating a composite location variable from $chromosome and $position and storing the alignment information in a temporary hash table
|
|
1128 ### extracting the chromosome number from the bowtie output (see above)
|
|
1129 my $alignment_location = join (":",$chromosome,$position);
|
|
1130 ### In the special case that two differently converted sequences align against differently converted genomes, but to the same position
|
|
1131 ### with the same number of mismatches (or perfect matches), the chromosome, position and number of mismatches are the same. In this
|
|
1132 ### case we are not writing the same entry out a second time.
|
|
1133 unless (exists $mismatches{$number_of_mismatches}->{$alignment_location}){
|
|
1134 $mismatches{$number_of_mismatches}->{$alignment_location}->{seq_id}=$id;
|
|
1135 $mismatches{$number_of_mismatches}->{$alignment_location}->{bowtie_sequence}=$bowtie_sequence;
|
|
1136 $mismatches{$number_of_mismatches}->{$alignment_location}->{index}=$index;
|
|
1137 $mismatches{$number_of_mismatches}->{$alignment_location}->{chromosome}=$chromosome;
|
|
1138 $mismatches{$number_of_mismatches}->{$alignment_location}->{position}=$position;
|
|
1139 }
|
|
1140 ####################################################################################################################################
|
|
1141 #### STEP III Now reading in one more line which has to be the next alignment to be analysed. Adding it to @fhs ->{last_line} ###
|
|
1142 ####################################################################################################################################
|
|
1143 $newline = $fhs[$index]->{fh}-> getline();
|
|
1144 if ($newline){
|
|
1145 my ($seq_id) = split (/\t/,$newline);
|
|
1146 die "The same seq ID occurred more than twice in a row\n" if ($seq_id eq $identifier);
|
|
1147 $fhs[$index]->{last_seq_id} = $seq_id;
|
|
1148 $fhs[$index]->{last_line} = $newline;
|
|
1149 next;
|
|
1150 }
|
|
1151 else {
|
|
1152 # assigning undef to last_seq_id and last_line and jumping to the next index (end of bowtie output)
|
|
1153 $fhs[$index]->{last_seq_id} = undef;
|
|
1154 $fhs[$index]->{last_line} = undef;
|
|
1155 next;
|
|
1156 }
|
|
1157 ### still within the 2nd sequence in correct orientation found
|
|
1158 }
|
|
1159 ### still withing the 1st sequence in correct orientation found
|
|
1160 }
|
|
1161 ### still within the if (last_seq_id eq identifier) condition
|
|
1162 }
|
|
1163 ### still within foreach index loop
|
|
1164 }
|
|
1165 ### if there was not a single alignment found for a certain sequence we will continue with the next sequence in the sequence file
|
|
1166 unless(%mismatches){
|
|
1167 $counting{no_single_alignment_found}++;
|
|
1168 if ($unmapped){
|
|
1169 return 1; ### We will print this sequence out as unmapped sequence if --un unmapped.out has been specified
|
|
1170 }
|
|
1171 else{
|
|
1172 return;
|
|
1173 }
|
|
1174 }
|
|
1175 #######################################################################################################################################################
|
|
1176 #######################################################################################################################################################
|
|
1177 ### We are now looking if there is a unique best alignment for a certain sequence. This means we are sorting in ascending order and look at the ###
|
|
1178 ### sequence with the lowest amount of mismatches. If there is only one single best position we are going to store the alignment information in the ###
|
|
1179 ### meth_call variables, if there are multiple hits with the same amount of (lowest) mismatches we are discarding the sequence altogether ###
|
|
1180 #######################################################################################################################################################
|
|
1181 #######################################################################################################################################################
|
|
1182 ### Going to use the variable $sequence_fails as a 'memory' if a sequence could not be aligned uniquely (set to 1 then)
|
|
1183 my $sequence_fails = 0;
|
|
1184 ### Declaring an empty hash reference which will store all information we need for the methylation call
|
|
1185 my $methylation_call_params; # hash reference!
|
|
1186 ### sorting in ascending order
|
|
1187 foreach my $mismatch_number (sort {$a<=>$b} keys %mismatches){
|
|
1188
|
|
1189 ### if there is only 1 entry in the hash with the lowest number of mismatches we accept it as the best alignment
|
|
1190 if (scalar keys %{$mismatches{$mismatch_number}} == 1){
|
|
1191 for my $unique_best_alignment (keys %{$mismatches{$mismatch_number}}){
|
|
1192 $methylation_call_params->{$identifier}->{bowtie_sequence} = $mismatches{$mismatch_number}->{$unique_best_alignment}->{bowtie_sequence};
|
|
1193 $methylation_call_params->{$identifier}->{chromosome} = $mismatches{$mismatch_number}->{$unique_best_alignment}->{chromosome};
|
|
1194 $methylation_call_params->{$identifier}->{position} = $mismatches{$mismatch_number}->{$unique_best_alignment}->{position};
|
|
1195 $methylation_call_params->{$identifier}->{index} = $mismatches{$mismatch_number}->{$unique_best_alignment}->{index};
|
|
1196 $methylation_call_params->{$identifier}->{number_of_mismatches} = $mismatch_number;
|
|
1197 }
|
|
1198 }
|
|
1199 elsif (scalar keys %{$mismatches{$mismatch_number}} == 3){
|
|
1200 ### If there are 3 sequences with the same number of lowest mismatches we can discriminate 2 cases: (i) all 3 alignments are unique best hits and
|
|
1201 ### come from different alignments processes (== indices) or (ii) one sequence alignment (== index) will give a unique best alignment, whereas a
|
|
1202 ### second one will produce 2 (or potentially many) alignments for the same sequence but in a different conversion state or against a different genome
|
|
1203 ### version (or both). This becomes especially relevant for highly converted sequences in which all Cs have been converted to Ts in the bisulfite
|
|
1204 ### reaction. E.g.
|
|
1205 ### CAGTCACGCGCGCGCG will become
|
|
1206 ### TAGTTATGTGTGTGTG in the CT transformed version, which will ideally still give the correct alignment in the CT->CT alignment condition.
|
|
1207 ### If the same read will then become G->A transformed as well however, the resulting sequence will look differently and potentially behave
|
|
1208 ### differently in a GA->GA alignment and this depends on the methylation state of the original sequence!:
|
|
1209 ### G->A conversion:
|
|
1210 ### highly methylated: CAATCACACACACACA
|
|
1211 ### highly converted : TAATTATATATATATA <== this sequence has a reduced complexity (only 2 bases left and not 3), and it is more likely to produce
|
|
1212 ### an alignment with a low complexity genomic region than the one above. This would normally lead to the entire sequence being kicked out as the
|
|
1213 ### there will be 3 alignments with the same number of lowest mismatches!! This in turn means that highly methylated and thereby not converted
|
|
1214 ### sequences are more likely to pass the alignment step, thereby creating a bias for methylated reads compared to their non-methylated counterparts.
|
|
1215 ### We do not want any bias, whatsover. Therefore if we have 1 sequence producing a unique best alignment and the second and third conditions
|
|
1216 ### producing alignments only after performing an additional (theoretical) conversion we want to keep the best alignment with the lowest number of
|
|
1217 ### additional transliterations performed. Thus we want to have a look at the level of complexity of the sequences producing the alignment.
|
|
1218 ### In the above example the number of transliterations required to transform the actual sequence
|
|
1219 ### to the C->T version would be TAGTTATGTGTGTGTG -> TAGTTATGTGTGTGTG = 0; (assuming this gives the correct alignment)
|
|
1220 ### in the G->A case it would be TAGTTATGTGTGTGTG -> TAATTATATATATATA = 6; (assuming this gives multiple wrong alignments)
|
|
1221 ### if the sequence giving a unique best alignment required a lower number of transliterations than the second best sequence yielding alignments
|
|
1222 ### while requiring a much higher number of transliterations, we are going to accept the unique best alignment with the lowest number of performed
|
|
1223 ### transliterations. As a threshold which does scale we will start with the number of tranliterations of the lowest best match x 2 must still be
|
|
1224 ### smaller than the number of tranliterations of the second best sequence. Everything will be flagged with $sequence_fails = 1 and discarded.
|
|
1225 my @three_candidate_seqs;
|
|
1226 foreach my $composite_location (keys (%{$mismatches{$mismatch_number}}) ){
|
|
1227 my $transliterations_performed;
|
|
1228 if ($mismatches{$mismatch_number}->{$composite_location}->{index} == 0 or $mismatches{$mismatch_number}->{$composite_location}->{index} == 1){
|
|
1229 $transliterations_performed = determine_number_of_transliterations_performed($sequence,'CT');
|
|
1230 }
|
|
1231 elsif ($mismatches{$mismatch_number}->{$composite_location}->{index} == 2 or $mismatches{$mismatch_number}->{$composite_location}->{index} == 3){
|
|
1232 $transliterations_performed = determine_number_of_transliterations_performed($sequence,'GA');
|
|
1233 }
|
|
1234 else{
|
|
1235 die "unexpected index number range $!\n";
|
|
1236 }
|
|
1237 push @three_candidate_seqs,{
|
|
1238 index =>$mismatches{$mismatch_number}->{$composite_location}->{index},
|
|
1239 bowtie_sequence => $mismatches{$mismatch_number}->{$composite_location}->{bowtie_sequence},
|
|
1240 mismatch_number => $mismatch_number,
|
|
1241 chromosome => $mismatches{$mismatch_number}->{$composite_location}->{chromosome},
|
|
1242 position => $mismatches{$mismatch_number}->{$composite_location}->{position},
|
|
1243 seq_id => $mismatches{$mismatch_number}->{$composite_location}->{seq_id},
|
|
1244 transliterations_performed => $transliterations_performed,
|
|
1245 };
|
|
1246 }
|
|
1247 ### sorting in ascending order for the lowest number of transliterations performed
|
|
1248 @three_candidate_seqs = sort {$a->{transliterations_performed} <=> $b->{transliterations_performed}} @three_candidate_seqs;
|
|
1249 my $first_array_element = $three_candidate_seqs[0]->{transliterations_performed};
|
|
1250 my $second_array_element = $three_candidate_seqs[1]->{transliterations_performed};
|
|
1251 my $third_array_element = $three_candidate_seqs[2]->{transliterations_performed};
|
|
1252 # print "$first_array_element\t$second_array_element\t$third_array_element\n";
|
|
1253 if (($first_array_element*2) < $second_array_element){
|
|
1254 $counting{low_complexity_alignments_overruled_count}++;
|
|
1255 ### taking the index with the unique best hit and over ruling low complexity alignments with 2 hits
|
|
1256 $methylation_call_params->{$identifier}->{bowtie_sequence} = $three_candidate_seqs[0]->{bowtie_sequence};
|
|
1257 $methylation_call_params->{$identifier}->{chromosome} = $three_candidate_seqs[0]->{chromosome};
|
|
1258 $methylation_call_params->{$identifier}->{position} = $three_candidate_seqs[0]->{position};
|
|
1259 $methylation_call_params->{$identifier}->{index} = $three_candidate_seqs[0]->{index};
|
|
1260 $methylation_call_params->{$identifier}->{number_of_mismatches} = $mismatch_number;
|
|
1261 # print "Overruled low complexity alignments! Using $first_array_element and disregarding $second_array_element and $third_array_element\n";
|
|
1262 }
|
|
1263 else{
|
|
1264 $sequence_fails = 1;
|
|
1265 }
|
|
1266 }
|
|
1267 else{
|
|
1268 $sequence_fails = 1;
|
|
1269 }
|
|
1270 ### after processing the alignment with the lowest number of mismatches we exit
|
|
1271 last;
|
|
1272 }
|
|
1273 ### skipping the sequence completely if there were multiple alignments with the same amount of lowest mismatches found at different positions
|
|
1274 if ($sequence_fails == 1){
|
|
1275 $counting{unsuitable_sequence_count}++;
|
|
1276 if ($ambiguous){
|
|
1277 return 2; # => exits to next sequence, and prints it out to multiple_alignments.out if --ambiguous has been specified
|
|
1278 }
|
|
1279 if ($unmapped){
|
|
1280 return 1; # => exits to next sequence, and prints it out to unmapped.out if --un has been specified
|
|
1281 }
|
|
1282 else{
|
|
1283 return 0; # => exits to next sequence (default)
|
|
1284 }
|
|
1285 }
|
|
1286
|
|
1287 ### --DIRECTIONAL
|
|
1288 ### If the option --directional has been specified the user wants to consider only alignments to the original top strand or the original bottom strand. We will therefore
|
|
1289 ### discard all alignments to strands complementary to the original strands, as they should not exist in reality due to the library preparation protocol
|
|
1290 if ($directional){
|
|
1291 if ( ($methylation_call_params->{$identifier}->{index} == 2) or ($methylation_call_params->{$identifier}->{index} == 3) ){
|
|
1292 # warn "Alignment rejected! (index was: $methylation_call_params->{$identifier}->{index})\n";
|
|
1293 $counting{alignments_rejected_count}++;
|
|
1294 return 0;
|
|
1295 }
|
|
1296 }
|
|
1297
|
|
1298 ### If the sequence has not been rejected so far it will have a unique best alignment
|
|
1299 $counting{unique_best_alignment_count}++;
|
|
1300 extract_corresponding_genomic_sequence_single_end($identifier,$methylation_call_params);
|
|
1301 ### check test to see if the genomic sequence we extracted has the same length as the observed sequence+2, and only then we perform the methylation call
|
|
1302 if (length($methylation_call_params->{$identifier}->{unmodified_genomic_sequence}) != length($sequence)+2){
|
|
1303 warn "Chromosomal sequence could not be extracted for\t$identifier\t$methylation_call_params->{$identifier}->{chromosome}\t$methylation_call_params->{$identifier}->{position}\n";
|
|
1304 $counting{genomic_sequence_could_not_be_extracted_count}++;
|
|
1305 return 0;
|
|
1306 }
|
|
1307
|
|
1308 ### otherwise we are set to perform the actual methylation call
|
|
1309 $methylation_call_params->{$identifier}->{methylation_call} = methylation_call($identifier,$sequence,$methylation_call_params->{$identifier}->{unmodified_genomic_sequence},$methylation_call_params->{$identifier}->{read_conversion});
|
|
1310
|
|
1311 print_bisulfite_mapping_result_single_end($identifier,$sequence,$methylation_call_params,$quality_value);
|
|
1312 return 0; ## otherwise 1 will be returned by default, which would print the sequence to unmapped.out
|
|
1313 }
|
|
1314
|
|
1315 sub check_bowtie_results_single_end_bowtie2{
|
|
1316 my ($sequence,$identifier,$quality_value) = @_;
|
|
1317
|
|
1318 unless ($quality_value){ # FastA sequences get assigned a quality value of Phred 40 throughout
|
|
1319 $quality_value = 'I'x(length$sequence);
|
|
1320 }
|
|
1321
|
|
1322 # as of version Bowtie 2 2.0.0 beta7, when input reads are unpaired, Bowtie 2 no longer removes the trailing /1 or /2 from the read name.
|
|
1323 # $identifier =~ s/\/[1234567890]+$//; # some sequencers don't just have /1 or /2 at the end of read IDs
|
|
1324
|
|
1325 my $alignment_ambiguous = 0;
|
|
1326
|
|
1327 my %alignments = ();
|
|
1328
|
|
1329 ### reading from the Bowtie 2 output filehandles
|
|
1330 foreach my $index (0..$#fhs){
|
|
1331 # print "Index: $index\n";
|
|
1332 # print "$fhs[$index]->{last_line}\n";
|
|
1333 # print "$fhs[$index]->{last_seq_id}\n\n";
|
|
1334
|
|
1335 ### skipping this index if the last alignment has been set to undefined already (i.e. end of bowtie output)
|
|
1336 next unless ($fhs[$index]->{last_line} and defined $fhs[$index]->{last_seq_id});
|
|
1337
|
|
1338 ### if the sequence we are currently looking at produced an alignment we are doing various things with it
|
|
1339 # print "last seq id: $fhs[$index]->{last_seq_id} and identifier: $identifier\n";
|
|
1340
|
|
1341 if ($fhs[$index]->{last_seq_id} eq $identifier) {
|
|
1342
|
|
1343 # SAM format specifications for Bowtie 2
|
|
1344 # (1) Name of read that aligned
|
|
1345 # (2) Sum of all applicable flags. Flags relevant to Bowtie are:
|
|
1346 # 1 The read is one of a pair
|
|
1347 # 2 The alignment is one end of a proper paired-end alignment
|
|
1348 # 4 The read has no reported alignments
|
|
1349 # 8 The read is one of a pair and has no reported alignments
|
|
1350 # 16 The alignment is to the reverse reference strand
|
|
1351 # 32 The other mate in the paired-end alignment is aligned to the reverse reference strand
|
|
1352 # 64 The read is mate 1 in a pair
|
|
1353 # 128 The read is mate 2 in a pair
|
|
1354 # 256 The read has multiple mapping states
|
|
1355 # (3) Name of reference sequence where alignment occurs (unmapped reads have a *)
|
|
1356 # (4) 1-based offset into the forward reference strand where leftmost character of the alignment occurs (0 for unmapped reads)
|
|
1357 # (5) Mapping quality (255 means MAPQ is not available)
|
|
1358 # (6) CIGAR string representation of alignment (* if unavailable)
|
|
1359 # (7) Name of reference sequence where mate's alignment occurs. Set to = if the mate's reference sequence is the same as this alignment's, or * if there is no mate.
|
|
1360 # (8) 1-based offset into the forward reference strand where leftmost character of the mate's alignment occurs. Offset is 0 if there is no mate.
|
|
1361 # (9) Inferred fragment size. Size is negative if the mate's alignment occurs upstream of this alignment. Size is 0 if there is no mate.
|
|
1362 # (10) Read sequence (reverse-complemented if aligned to the reverse strand)
|
|
1363 # (11) ASCII-encoded read qualities (reverse-complemented if the read aligned to the reverse strand). The encoded quality values are on the Phred quality scale and the encoding is ASCII-offset by 33 (ASCII char !), similarly to a FASTQ file.
|
|
1364 # (12) Optional fields. Fields are tab-separated. bowtie2 outputs zero or more of these optional fields for each alignment, depending on the type of the alignment:
|
|
1365 # AS:i:<N> Alignment score. Can be negative. Can be greater than 0 in --local mode (but not in --end-to-end mode). Only present if SAM record is for an aligned read.
|
|
1366 # XS:i:<N> Alignment score for second-best alignment. Can be negative. Can be greater than 0 in --local mode (but not in --end-to-end mode). Only present if the SAM record is for an aligned read and more than one alignment was found for the read.
|
|
1367 # YS:i:<N> Alignment score for opposite mate in the paired-end alignment. Only present if the SAM record is for a read that aligned as part of a paired-end alignment.
|
|
1368 # XN:i:<N> The number of ambiguous bases in the reference covering this alignment. Only present if SAM record is for an aligned read.
|
|
1369 # XM:i:<N> The number of mismatches in the alignment. Only present if SAM record is for an aligned read.
|
|
1370 # XO:i:<N> The number of gap opens, for both read and reference gaps, in the alignment. Only present if SAM record is for an aligned read.
|
|
1371 # XG:i:<N> The number of gap extensions, for both read and reference gaps, in the alignment. Only present if SAM record is for an aligned read.
|
|
1372 # NM:i:<N> The edit distance; that is, the minimal number of one-nucleotide edits (substitutions, insertions and deletions) needed to transform the read string into the reference string. Only present if SAM record is for an aligned read.
|
|
1373 # YF:Z:<N> String indicating reason why the read was filtered out. See also: Filtering. Only appears for reads that were filtered out.
|
|
1374 # MD:Z:<S> A string representation of the mismatched reference bases in the alignment. See SAM format specification for details. Only present if SAM record is for an aligned read.
|
|
1375
|
|
1376 my ($id,$flag,$mapped_chromosome,$position,$mapping_quality,$cigar,$bowtie_sequence,$qual) = (split (/\t/,$fhs[$index]->{last_line}))[0,1,2,3,4,5,9,10];
|
|
1377
|
|
1378 ### If a sequence has no reported alignments there will be a single output line with a bit-wise flag value of 4. We can store the next alignment and move on to the next Bowtie 2 instance
|
|
1379 if ($flag == 4){
|
|
1380 ## reading in the next alignment, which must be the next sequence
|
|
1381 my $newline = $fhs[$index]->{fh}-> getline();
|
|
1382 if ($newline){
|
|
1383 chomp $newline;
|
|
1384 my ($seq_id) = split (/\t/,$newline);
|
|
1385 $fhs[$index]->{last_seq_id} = $seq_id;
|
|
1386 $fhs[$index]->{last_line} = $newline;
|
|
1387 if ($seq_id eq $identifier){
|
|
1388 die "Sequence with ID $identifier did not produce any alignment, but next seq-ID was also $fhs[$index]->{last_seq_id}!\n";
|
|
1389 }
|
|
1390 next; # next instance
|
|
1391 }
|
|
1392 else{
|
|
1393 # assigning undef to last_seq_id and last_line and jumping to the next index (end of Bowtie 2 output)
|
|
1394 $fhs[$index]->{last_seq_id} = undef;
|
|
1395 $fhs[$index]->{last_line} = undef;
|
|
1396 next;
|
|
1397 }
|
|
1398 }
|
|
1399
|
|
1400 # if there are one or more proper alignments we can extract the chromosome number
|
|
1401 my $chromosome;
|
|
1402 if ($mapped_chromosome =~ s/_(CT|GA)_converted$//){
|
|
1403 $chromosome = $mapped_chromosome;
|
|
1404 }
|
|
1405 else{
|
|
1406 die "Chromosome number extraction failed for $mapped_chromosome\n";
|
|
1407 }
|
|
1408
|
|
1409 ### We will use the optional field to determine the best alignment. Later on we extract the number of mismatches and/or indels from the CIGAR string
|
|
1410 my ($alignment_score,$second_best,$MD_tag);
|
|
1411 my @fields = split (/\t/,$fhs[$index]->{last_line});
|
|
1412
|
|
1413 foreach (11..$#fields){
|
|
1414 if ($fields[$_] =~ /AS:i:(.*)/){
|
|
1415 $alignment_score = $1;
|
|
1416 }
|
|
1417 elsif ($fields[$_] =~ /XS:i:(.*)/){
|
|
1418 $second_best = $1;
|
|
1419 }
|
|
1420 elsif ($fields[$_] =~ /MD:Z:(.*)/){
|
|
1421 $MD_tag = $1;
|
|
1422 }
|
|
1423 }
|
|
1424
|
|
1425 # warn "First best alignment_score is: '$alignment_score'\n";
|
|
1426 # warn "MD tag is: '$MD_tag'\n";
|
|
1427 die "Failed to extract alignment score ($alignment_score) and MD tag ($MD_tag)!\n" unless (defined $alignment_score and defined $MD_tag);
|
|
1428
|
|
1429 if (defined $second_best){
|
|
1430 # warn "second best alignment_score is: '$second_best'\n";
|
|
1431
|
|
1432 # If the first alignment score is the same as the alignment score of the second best hit we are going to boot this sequence altogether
|
|
1433 if ($alignment_score == $second_best){
|
|
1434 $alignment_ambiguous = 1;
|
|
1435 ## need to read and discard all additional ambiguous reads until we reach the next sequence
|
|
1436 until ($fhs[$index]->{last_seq_id} ne $identifier){
|
|
1437 my $newline = $fhs[$index]->{fh}-> getline();
|
|
1438 if ($newline){
|
|
1439 chomp $newline;
|
|
1440 my ($seq_id) = split (/\t/,$newline);
|
|
1441 $fhs[$index]->{last_seq_id} = $seq_id;
|
|
1442 $fhs[$index]->{last_line} = $newline;
|
|
1443 }
|
|
1444 else{
|
|
1445 # assigning undef to last_seq_id and last_line and jumping to the next index (end of Bowtie 2 output)
|
|
1446 $fhs[$index]->{last_seq_id} = undef;
|
|
1447 $fhs[$index]->{last_line} = undef;
|
|
1448 last; # break free in case we have reached the end of the alignment output
|
|
1449 }
|
|
1450 }
|
|
1451 # warn "Index: $index\tThe current Seq-ID is $identifier, skipped all ambiguous sequences until the next ID which is: $fhs[$index]->{last_seq_id}\n";
|
|
1452 }
|
|
1453 else{ # the next best alignment has a lower alignment score than the current read, so we can safely store the current alignment
|
|
1454
|
|
1455 my $alignment_location = join (":",$chromosome,$position);
|
|
1456
|
|
1457 ### If a sequence aligns to exactly the same location with a perfect match twice the sequence does either not contain any C or G, or all the Cs (or Gs on the reverse
|
|
1458 ### strand) were methylated and therefore protected. Alternatively it will align better in one condition than in the other. In any case, it is not needed to overwrite
|
|
1459 ### the same positional entry with a second entry for the same location, as the genomic sequence extraction and methylation call would not be affected by this. The only
|
|
1460 ### thing which would change is the index number for the found alignment). We will continue to assign these alignments to the first indexes 0 and 1, i.e. OT and OB
|
|
1461
|
|
1462 unless (exists $alignments{$alignment_location}){
|
|
1463 $alignments{$alignment_location}->{seq_id} = $id;
|
|
1464 $alignments{$alignment_location}->{alignment_score} = $alignment_score;
|
|
1465 $alignments{$alignment_location}->{bowtie_sequence} = $bowtie_sequence;
|
|
1466 $alignments{$alignment_location}->{index} = $index;
|
|
1467 $alignments{$alignment_location}->{chromosome} = $chromosome;
|
|
1468 $alignments{$alignment_location}->{position} = $position;
|
|
1469 $alignments{$alignment_location}->{CIGAR} = $cigar;
|
|
1470 $alignments{$alignment_location}->{MD_tag} = $MD_tag;
|
|
1471 }
|
|
1472
|
|
1473 ### now reading and discarding all (inferior) alignments of this sequencing read until we hit the next sequence
|
|
1474 until ($fhs[$index]->{last_seq_id} ne $identifier){
|
|
1475 my $newline = $fhs[$index]->{fh}-> getline();
|
|
1476 if ($newline){
|
|
1477 chomp $newline;
|
|
1478 my ($seq_id) = split (/\t/,$newline);
|
|
1479 $fhs[$index]->{last_seq_id} = $seq_id;
|
|
1480 $fhs[$index]->{last_line} = $newline;
|
|
1481 }
|
|
1482 else{
|
|
1483 # assigning undef to last_seq_id and last_line and jumping to the next index (end of Bowtie 2 output)
|
|
1484 $fhs[$index]->{last_seq_id} = undef;
|
|
1485 $fhs[$index]->{last_line} = undef;
|
|
1486 last; # break free in case we have reached the end of the alignment output
|
|
1487 }
|
|
1488 }
|
|
1489 # warn "Index: $index\tThe current Seq-ID is $identifier, skipped all ambiguous sequences until the next ID which is: $fhs[$index]->{last_seq_id}\n";
|
|
1490 }
|
|
1491 }
|
|
1492 else{ # there is no second best hit, so we can just store this one and read in the next sequence
|
|
1493
|
|
1494 my $alignment_location = join (":",$chromosome,$position);
|
|
1495
|
|
1496 ### If a sequence aligns to exactly the same location with a perfect match twice the sequence does either not contain any C or G, or all the Cs (or Gs on the reverse
|
|
1497 ### strand) were methylated and therefore protected. Alternatively it will align better in one condition than in the other. In any case, it is not needed to overwrite
|
|
1498 ### the same positional entry with a second entry for the same location, as the genomic sequence extraction and methylation call would not be affected by this. The only
|
|
1499 ### thing which would change is the index number for the found alignment). We will continue to assign these alignments to the first indexes 0 and 1, i.e. OT and OB
|
|
1500
|
|
1501 unless (exists $alignments{$alignment_location}){
|
|
1502 $alignments{$alignment_location}->{seq_id} = $id;
|
|
1503 $alignments{$alignment_location}->{alignment_score} = $alignment_score;
|
|
1504 $alignments{$alignment_location}->{bowtie_sequence} = $bowtie_sequence;
|
|
1505 $alignments{$alignment_location}->{index} = $index;
|
|
1506 $alignments{$alignment_location}->{chromosome} = $chromosome;
|
|
1507 $alignments{$alignment_location}->{position} = $position;
|
|
1508 $alignments{$alignment_location}->{MD_tag} = $MD_tag;
|
|
1509 $alignments{$alignment_location}->{CIGAR} = $cigar;
|
|
1510 }
|
|
1511
|
|
1512 my $newline = $fhs[$index]->{fh}-> getline();
|
|
1513 if ($newline){
|
|
1514 chomp $newline;
|
|
1515 my ($seq_id) = split (/\t/,$newline);
|
|
1516 $fhs[$index]->{last_seq_id} = $seq_id;
|
|
1517 $fhs[$index]->{last_line} = $newline;
|
|
1518 if ($seq_id eq $identifier){
|
|
1519 die "Sequence with ID $identifier did not have a second best alignment, but next seq-ID was also $fhs[$index]->{last_seq_id}!\n";
|
|
1520 }
|
|
1521 }
|
|
1522 else{
|
|
1523 # assigning undef to last_seq_id and last_line and jumping to the next index (end of Bowtie 2 output)
|
|
1524 $fhs[$index]->{last_seq_id} = undef;
|
|
1525 $fhs[$index]->{last_line} = undef;
|
|
1526 }
|
|
1527 }
|
|
1528 }
|
|
1529 }
|
|
1530
|
|
1531 ### if the read produced several ambiguous alignments already now can returning already now. If --ambiguous or --unmapped was specified the read sequence will be printed out.
|
|
1532 if ($alignment_ambiguous == 1){
|
|
1533 $counting{unsuitable_sequence_count}++;
|
|
1534 ### report that the sequence has multiple hits with bitwise flag 256. We can print the sequence to the result file straight away and skip everything else
|
|
1535 # my $ambiguous_read_output = join("\t",$identifier,'256','*','0','0','*','*','0','0',$sequence,$quality_value);
|
|
1536 # print "$ambiguous_read_output\n";
|
|
1537
|
|
1538 if ($ambiguous){
|
|
1539 return 2; # => exits to next sequence, and prints it out to _ambiguous_reads.txt if '--ambiguous' was specified
|
|
1540 }
|
|
1541 elsif ($unmapped){
|
|
1542 return 1; # => exits to next sequence, and prints it out to _unmapped_reads.txt if '--unmapped' but not '--ambiguous' was specified
|
|
1543 }
|
|
1544 else{
|
|
1545 return 0;
|
|
1546 }
|
|
1547 }
|
|
1548
|
|
1549 ### if there was no alignment found for a certain sequence at all we continue with the next sequence in the sequence file
|
|
1550 unless(%alignments){
|
|
1551 $counting{no_single_alignment_found}++;
|
|
1552 # my $unmapped_read_output = join("\t",$identifier,'4','*','0','0','*','*','0','0',$sequence,$quality_value);
|
|
1553 # print "$unmapped_read_output\n";
|
|
1554 if ($unmapped){
|
|
1555 return 1; # => exits to next sequence, and prints it out to _unmapped_reads.txt if '--unmapped' was specified
|
|
1556 }
|
|
1557 else{
|
|
1558 return 0; # default
|
|
1559 }
|
|
1560 }
|
|
1561
|
|
1562 #######################################################################################################################################################
|
|
1563
|
|
1564 ### If the sequence was not rejected so far we are now looking if there is a unique best alignment among all alignment instances. If there is only one
|
|
1565 ### single best position we are going to store the alignment information in the $meth_call variable. If there are multiple hits with the same (highest)
|
|
1566 ### alignment score we are discarding the sequence altogether.
|
|
1567 ### For end-to-end alignments the maximum alignment score can be 0, each mismatch can receive penalties up to 6, and each gap receives penalties for
|
|
1568 ### opening (5) and extending (3 per bp) the gap.
|
|
1569
|
|
1570 #######################################################################################################################################################
|
|
1571
|
|
1572 my $methylation_call_params; # hash reference which will store all information we need for the methylation call
|
|
1573 my $sequence_fails = 0; # Going to use $sequence_fails as a 'memory' if a sequence could not be aligned uniquely (set to 1 then)
|
|
1574
|
|
1575 ### print contents of %alignments for debugging
|
|
1576 # if (scalar keys %alignments > 1){
|
|
1577 # print "\n******\n";
|
|
1578 # foreach my $alignment_location (sort {$a cmp $b} keys %alignments){
|
|
1579 # print "Loc: $alignment_location\n";
|
|
1580 # print "ID: $alignments{$alignment_location}->{seq_id}\n";
|
|
1581 # print "AS: $alignments{$alignment_location}->{alignment_score}\n";
|
|
1582 # print "Seq: $alignments{$alignment_location}->{bowtie_sequence}\n";
|
|
1583 # print "Index $alignments{$alignment_location}->{index}\n";
|
|
1584 # print "Chr: $alignments{$alignment_location}->{chromosome}\n";
|
|
1585 # print "pos: $alignments{$alignment_location}->{position}\n";
|
|
1586 # print "MD: $alignments{$alignment_location}->{MD_tag}\n\n";
|
|
1587 # }
|
|
1588 # print "\n******\n";
|
|
1589 # }
|
|
1590
|
|
1591 ### if there is only 1 entry in the hash with we accept it as the best alignment
|
|
1592 if (scalar keys %alignments == 1){
|
|
1593 for my $unique_best_alignment (keys %alignments){
|
|
1594 $methylation_call_params->{$identifier}->{bowtie_sequence} = $alignments{$unique_best_alignment}->{bowtie_sequence};
|
|
1595 $methylation_call_params->{$identifier}->{chromosome} = $alignments{$unique_best_alignment}->{chromosome};
|
|
1596 $methylation_call_params->{$identifier}->{position} = $alignments{$unique_best_alignment}->{position};
|
|
1597 $methylation_call_params->{$identifier}->{index} = $alignments{$unique_best_alignment}->{index};
|
|
1598 $methylation_call_params->{$identifier}->{alignment_score} = $alignments{$unique_best_alignment}->{alignment_score};
|
|
1599 $methylation_call_params->{$identifier}->{MD_tag} = $alignments{$unique_best_alignment}->{MD_tag};
|
|
1600 $methylation_call_params->{$identifier}->{CIGAR} = $alignments{$unique_best_alignment}->{CIGAR};
|
|
1601 }
|
|
1602 }
|
|
1603
|
|
1604 ### otherwise we are going to find out if there is a best match among the multiple alignments, or whether there are 2 or more equally good alignments (in which case
|
|
1605 ### we boot the sequence altogether
|
|
1606 elsif (scalar keys %alignments >= 2 and scalar keys %alignments <= 4){
|
|
1607 my $best_alignment_score;
|
|
1608 my $best_alignment_location;
|
|
1609 foreach my $alignment_location (sort {$alignments{$b}->{alignment_score} <=> $alignments{$a}->{alignment_score}} keys %alignments){
|
|
1610 # print "$alignments{$alignment_location}->{alignment_score}\n";
|
|
1611 unless (defined $best_alignment_score){
|
|
1612 $best_alignment_score = $alignments{$alignment_location}->{alignment_score};
|
|
1613 $best_alignment_location = $alignment_location;
|
|
1614 # print "setting best alignment score: $best_alignment_score\n";
|
|
1615 }
|
|
1616 else{
|
|
1617 ### if the second best alignment has the same alignment score as the first one, the sequence will get booted
|
|
1618 if ($alignments{$alignment_location}->{alignment_score} == $best_alignment_score){
|
|
1619 # warn "Same alignment score, the sequence will get booted!\n";
|
|
1620 $sequence_fails = 1;
|
|
1621 last; # exiting after the second alignment since we know that the sequence has ambiguous alignments
|
|
1622 }
|
|
1623 ### else we are going to store the best alignment for further processing
|
|
1624 else{
|
|
1625 $methylation_call_params->{$identifier}->{bowtie_sequence} = $alignments{$best_alignment_location}->{bowtie_sequence};
|
|
1626 $methylation_call_params->{$identifier}->{chromosome} = $alignments{$best_alignment_location}->{chromosome};
|
|
1627 $methylation_call_params->{$identifier}->{position} = $alignments{$best_alignment_location}->{position};
|
|
1628 $methylation_call_params->{$identifier}->{index} = $alignments{$best_alignment_location}->{index};
|
|
1629 $methylation_call_params->{$identifier}->{alignment_score} = $alignments{$best_alignment_location}->{alignment_score};
|
|
1630 $methylation_call_params->{$identifier}->{MD_tag} = $alignments{$best_alignment_location}->{MD_tag};
|
|
1631 $methylation_call_params->{$identifier}->{CIGAR} = $alignments{$best_alignment_location}->{CIGAR};
|
|
1632 last; # exiting after processing the second alignment since the sequence produced a unique best alignment
|
|
1633 }
|
|
1634 }
|
|
1635 }
|
|
1636 }
|
|
1637 else{
|
|
1638 die "There are too many potential hits for this sequence (1-4 expected, but found: ",scalar keys %alignments,")\n";;
|
|
1639 }
|
|
1640
|
|
1641 ### skipping the sequence completely if there were multiple alignments with the same best alignment score at different positions
|
|
1642 if ($sequence_fails == 1){
|
|
1643 $counting{unsuitable_sequence_count}++;
|
|
1644
|
|
1645 ### report that the sequence has multiple hits with bitwise flag 256. We can print the sequence to the result file straight away and skip everything else
|
|
1646 # my $ambiguous_read_output = join("\t",$identifier,'256','*','0','0','*','*','0','0',$sequence,$quality_value);
|
|
1647 # print OUT "$ambiguous_read_output\n";
|
|
1648
|
|
1649 if ($ambiguous){
|
|
1650 return 2; # => exits to next sequence, and prints it out (in FastQ format) to _ambiguous_reads.txt if '--ambiguous' was specified
|
|
1651 }
|
|
1652 elsif ($unmapped){
|
|
1653 return 1; # => exits to next sequence, and prints it out (in FastQ format) to _unmapped_reads.txt if '--unmapped' but not '--ambiguous' was specified
|
|
1654 }
|
|
1655 else{
|
|
1656 return 0; # => exits to next sequence (default)
|
|
1657 }
|
|
1658 }
|
|
1659
|
|
1660 ### --DIRECTIONAL
|
|
1661 ### If the option --directional has been specified the user wants to consider only alignments to the original top strand or the original bottom strand. We will therefore
|
|
1662 ### discard all alignments to strands complementary to the original strands, as they should not exist in reality due to the library preparation protocol
|
|
1663 if ($directional){
|
|
1664 if ( ($methylation_call_params->{$identifier}->{index} == 2) or ($methylation_call_params->{$identifier}->{index} == 3) ){
|
|
1665 # warn "Alignment rejected! (index was: $methylation_call_params->{$identifier}->{index})\n";
|
|
1666 $counting{alignments_rejected_count}++;
|
|
1667 return 0;
|
|
1668 }
|
|
1669 }
|
|
1670
|
|
1671 ### If the sequence has not been rejected so far it has a unique best alignment
|
|
1672 $counting{unique_best_alignment_count}++;
|
|
1673
|
|
1674 ### Now we need to extract a genomic sequence that exactly corresponds to the reported alignment. This potentially means that we need to deal with insertions or deletions as well
|
|
1675 extract_corresponding_genomic_sequence_single_end_bowtie2 ($identifier,$methylation_call_params);
|
|
1676
|
|
1677 ### check test to see if the genomic sequence we extracted has the same length as the observed sequence+2, and only then we perform the methylation call
|
|
1678 if (length($methylation_call_params->{$identifier}->{unmodified_genomic_sequence}) != length($sequence)+2){
|
|
1679 warn "Chromosomal sequence could not be extracted for\t$identifier\t$methylation_call_params->{$identifier}->{chromosome}\t$methylation_call_params->{$identifier}->{position}\n";
|
|
1680 $counting{genomic_sequence_could_not_be_extracted_count}++;
|
|
1681 return 0;
|
|
1682 }
|
|
1683
|
|
1684
|
|
1685 ### otherwise we are set to perform the actual methylation call
|
|
1686 $methylation_call_params->{$identifier}->{methylation_call} = methylation_call($identifier,$sequence,$methylation_call_params->{$identifier}->{unmodified_genomic_sequence},$methylation_call_params->{$identifier}->{read_conversion});
|
|
1687 print_bisulfite_mapping_result_single_end_bowtie2 ($identifier,$sequence,$methylation_call_params,$quality_value);
|
|
1688 return 0; ## if a sequence got this far we do not want to print it to unmapped or ambiguous.out
|
|
1689 }
|
|
1690
|
|
1691
|
|
1692 sub determine_number_of_transliterations_performed{
|
|
1693 my ($sequence,$read_conversion) = @_;
|
|
1694 my $number_of_transliterations;
|
|
1695 if ($read_conversion eq 'CT'){
|
|
1696 $number_of_transliterations = $sequence =~ tr/C/T/;
|
|
1697 }
|
|
1698 elsif ($read_conversion eq 'GA'){
|
|
1699 $number_of_transliterations = $sequence =~ tr/G/A/;
|
|
1700 }
|
|
1701 else{
|
|
1702 die "Read conversion mode of the read was not specified $!\n";
|
|
1703 }
|
|
1704 return $number_of_transliterations;
|
|
1705 }
|
|
1706
|
|
1707 sub decide_whether_single_end_alignment_is_valid{
|
|
1708 my ($index,$identifier) = @_;
|
|
1709
|
|
1710 # extracting from Bowtie 1 format
|
|
1711 my ($id,$strand) = (split (/\t/,$fhs[$index]->{last_line}))[0,1];
|
|
1712
|
|
1713 ### ensuring that the entry is the correct sequence
|
|
1714 if (($id eq $fhs[$index]->{last_seq_id}) and ($id eq $identifier)){
|
|
1715 ### checking the orientation of the alignment. We need to discriminate between 8 different conditions, however only 4 of them are theoretically
|
|
1716 ### sensible alignments
|
|
1717 my $orientation = ensure_sensical_alignment_orientation_single_end ($index,$strand);
|
|
1718 ### If the orientation was correct can we move on
|
|
1719 if ($orientation == 1){
|
|
1720 return 1; ### 1st possibility for a sequence to pass
|
|
1721 }
|
|
1722 ### If the alignment was in the wrong orientation we need to read in a new line
|
|
1723 elsif($orientation == 0){
|
|
1724 my $newline = $fhs[$index]->{fh}->getline();
|
|
1725 if ($newline){
|
|
1726 ($id,$strand) = (split (/\t/,$newline))[0,1];
|
|
1727
|
|
1728 ### ensuring that the next entry is still the correct sequence
|
|
1729 if ($id eq $identifier){
|
|
1730 ### checking orientation again
|
|
1731 $orientation = ensure_sensical_alignment_orientation_single_end ($index,$strand);
|
|
1732 ### If the orientation was correct can we move on
|
|
1733 if ($orientation == 1){
|
|
1734 $fhs[$index]->{last_seq_id} = $id;
|
|
1735 $fhs[$index]->{last_line} = $newline;
|
|
1736 return 1; ### 2nd possibility for a sequence to pass
|
|
1737 }
|
|
1738 ### If the alignment was in the wrong orientation again we need to read in yet another new line and store it in @fhs
|
|
1739 elsif ($orientation == 0){
|
|
1740 $newline = $fhs[$index]->{fh}->getline();
|
|
1741 if ($newline){
|
|
1742 my ($seq_id) = split (/\t/,$newline);
|
|
1743 ### check if the next line still has the same seq ID (must not happen), and if not overwrite the current seq-ID and bowtie output with
|
|
1744 ### the same fields of the just read next entry
|
|
1745 die "Same seq ID 3 or more times in a row!(should be 2 max) $!" if ($seq_id eq $identifier);
|
|
1746 $fhs[$index]->{last_seq_id} = $seq_id;
|
|
1747 $fhs[$index]->{last_line} = $newline;
|
|
1748 return 0; # not processing anything this round as the alignment currently stored in last_line was in the wrong orientation
|
|
1749 }
|
|
1750 else{
|
|
1751 # assigning undef to last_seq_id and last_line (end of bowtie output)
|
|
1752 $fhs[$index]->{last_seq_id} = undef;
|
|
1753 $fhs[$index]->{last_line} = undef;
|
|
1754 return 0; # not processing anything as the alignment currently stored in last_line was in the wrong orientation
|
|
1755 }
|
|
1756 }
|
|
1757 else{
|
|
1758 die "The orientation of the alignment must be either correct or incorrect\n";
|
|
1759 }
|
|
1760 }
|
|
1761 ### the sequence we just read in is already the next sequence to be analysed -> store it in @fhs
|
|
1762 else{
|
|
1763 $fhs[$index]->{last_seq_id} = $id;
|
|
1764 $fhs[$index]->{last_line} = $newline;
|
|
1765 return 0; # processing the new alignment result only in the next round
|
|
1766 }
|
|
1767 }
|
|
1768 else {
|
|
1769 # assigning undef to last_seq_id and last_line (end of bowtie output)
|
|
1770 $fhs[$index]->{last_seq_id} = undef;
|
|
1771 $fhs[$index]->{last_line} = undef;
|
|
1772 return 0; # not processing anything as the alignment currently stored in last_line was in the wrong orientation
|
|
1773 }
|
|
1774 }
|
|
1775 else{
|
|
1776 die "The orientation of the alignment must be either correct or incorrect\n";
|
|
1777 }
|
|
1778 }
|
|
1779 ### the sequence stored in @fhs as last_line is already the next sequence to be analysed -> analyse next round
|
|
1780 else{
|
|
1781 return 0;
|
|
1782 }
|
|
1783 }
|
|
1784 #########################
|
|
1785 ### BOWTIE 1 | PAIRED-END
|
|
1786 #########################
|
|
1787
|
|
1788 sub check_bowtie_results_paired_ends{
|
|
1789 my ($sequence_1,$sequence_2,$identifier,$quality_value_1,$quality_value_2) = @_;
|
|
1790
|
|
1791 ### quality values are not given for FastA files, so they are initialised with a Phred quality of 40
|
|
1792 unless ($quality_value_1){
|
|
1793 $quality_value_1 = 'I'x(length$sequence_1);
|
|
1794 }
|
|
1795 unless ($quality_value_2){
|
|
1796 $quality_value_2 = 'I'x(length$sequence_2);
|
|
1797 }
|
|
1798
|
|
1799 # print "$identifier\n$fhs[0]->{last_seq_id}\n$fhs[1]->{last_seq_id}\n$fhs[2]->{last_seq_id}\n$fhs[3]->{last_seq_id}\n\n";
|
|
1800
|
|
1801 my %mismatches = ();
|
|
1802 ### reading from the bowtie output files to see if this sequence pair aligned to a bisulfite converted genome
|
|
1803
|
|
1804
|
|
1805 ### for paired end reads we are reporting alignments to the OT strand first (index 0), then the OB strand (index 3!!), similiar to the single end way.
|
|
1806 ### alignments to the complementary strands are reported afterwards (CTOT got index 1, and CTOB got index 2).
|
|
1807 ### This is needed so that alignments which either contain no single C or G or reads which contain only protected Cs are reported to the original strands (OT and OB)
|
|
1808 ### Before the complementary strands. Remember that it does not make any difference for the methylation calls, but it will matter if alignment to the complementary
|
|
1809 ### strands are not being reported by specifying --directional
|
|
1810
|
|
1811 foreach my $index (0,3,1,2){
|
|
1812 ### skipping this index if the last alignment has been set to undefined already (i.e. end of bowtie output)
|
|
1813 next unless ($fhs[$index]->{last_line_1} and $fhs[$index]->{last_line_2} and defined $fhs[$index]->{last_seq_id});
|
|
1814 ### if the sequence pair we are currently looking at produced an alignment we are doing various things with it
|
|
1815 if ($fhs[$index]->{last_seq_id} eq $identifier) {
|
|
1816 # print "$identifier\n$fhs[$index]->{last_seq_id}\n\n";
|
|
1817
|
|
1818 ##################################################################################
|
|
1819 ### STEP I Processing the entry which is stored in last_line_1 and last_line_2 ###
|
|
1820 ##################################################################################
|
|
1821 my $valid_alignment_found = decide_whether_paired_end_alignment_is_valid($index,$identifier);
|
|
1822 ### sequences can fail at this point if there was only 1 alignment in the wrong orientation, or if there were 2 aligments both in the wrong
|
|
1823 ### orientation. We only continue to extract useful information about this alignment if 1 was returned
|
|
1824 if ($valid_alignment_found == 1){
|
|
1825 ### Bowtie outputs which made it this far are in the correct orientation, so we can continue to analyse the alignment itself.
|
|
1826 ### we store the useful information in %mismatches
|
|
1827 my ($id_1,$strand_1,$mapped_chromosome_1,$position_1,$bowtie_sequence_1,$mismatch_info_1) = (split (/\t/,$fhs[$index]->{last_line_1},-1))[0,1,2,3,4,7];
|
|
1828 my ($id_2,$strand_2,$mapped_chromosome_2,$position_2,$bowtie_sequence_2,$mismatch_info_2) = (split (/\t/,$fhs[$index]->{last_line_2},-1))[0,1,2,3,4,7];
|
|
1829 chomp $mismatch_info_1;
|
|
1830 chomp $mismatch_info_2;
|
|
1831
|
|
1832 ### need to extract the chromosome number from the bowtie output (which is either XY_CT_converted or XY_GA_converted
|
|
1833 my ($chromosome_1,$chromosome_2);
|
|
1834 if ($mapped_chromosome_1 =~ s/_(CT|GA)_converted$//){
|
|
1835 $chromosome_1 = $mapped_chromosome_1;
|
|
1836 }
|
|
1837 else{
|
|
1838 die "Chromosome number extraction failed for $mapped_chromosome_1\n";
|
|
1839 }
|
|
1840 if ($mapped_chromosome_2 =~ s/_(CT|GA)_converted$//){
|
|
1841 $chromosome_2 = $mapped_chromosome_2;
|
|
1842 }
|
|
1843 else{
|
|
1844 die "Chromosome number extraction failed for $mapped_chromosome_2\n";
|
|
1845 }
|
|
1846
|
|
1847 ### Now extracting the number of mismatches to the converted genome
|
|
1848 my $number_of_mismatches_1;
|
|
1849 my $number_of_mismatches_2;
|
|
1850 if ($mismatch_info_1 eq ''){
|
|
1851 $number_of_mismatches_1 = 0;
|
|
1852 }
|
|
1853 elsif ($mismatch_info_1 =~ /^\d/){
|
|
1854 my @mismatches = split (/,/,$mismatch_info_1);
|
|
1855 $number_of_mismatches_1 = scalar @mismatches;
|
|
1856 }
|
|
1857 else{
|
|
1858 die "Something weird is going on with the mismatch field\n";
|
|
1859 }
|
|
1860 if ($mismatch_info_2 eq ''){
|
|
1861 $number_of_mismatches_2 = 0;
|
|
1862 }
|
|
1863 elsif ($mismatch_info_2 =~ /^\d/){
|
|
1864 my @mismatches = split (/,/,$mismatch_info_2);
|
|
1865 $number_of_mismatches_2 = scalar @mismatches;
|
|
1866 }
|
|
1867 else{
|
|
1868 die "Something weird is going on with the mismatch field\n";
|
|
1869 }
|
|
1870 ### To decide whether a sequence pair has a unique best alignment we will look at the lowest sum of mismatches from both alignments
|
|
1871 my $sum_of_mismatches = $number_of_mismatches_1+$number_of_mismatches_2;
|
|
1872 ### creating a composite location variable from $chromosome and $position and storing the alignment information in a temporary hash table
|
|
1873 die "Position 1 is higher than position 2" if ($position_1 > $position_2);
|
|
1874 die "Paired-end alignments need to be on the same chromosome\n" unless ($chromosome_1 eq $chromosome_2);
|
|
1875 my $alignment_location = join(":",$chromosome_1,$position_1,$position_2);
|
|
1876 ### If a sequence aligns to exactly the same location twice the sequence does either not contain any C or G, or all the Cs (or Gs on the reverse
|
|
1877 ### strand) were methylated and therefore protected. It is not needed to overwrite the same positional entry with a second entry for the same
|
|
1878 ### location (the genomic sequence extraction and methylation would not be affected by this, only the thing which would change is the index
|
|
1879 ### number for the found alignment)
|
|
1880 unless (exists $mismatches{$sum_of_mismatches}->{$alignment_location}){
|
|
1881 $mismatches{$sum_of_mismatches}->{$alignment_location}->{seq_id}=$id_1; # either is fine
|
|
1882 $mismatches{$sum_of_mismatches}->{$alignment_location}->{bowtie_sequence_1}=$bowtie_sequence_1;
|
|
1883 $mismatches{$sum_of_mismatches}->{$alignment_location}->{bowtie_sequence_2}=$bowtie_sequence_2;
|
|
1884 $mismatches{$sum_of_mismatches}->{$alignment_location}->{index}=$index;
|
|
1885 $mismatches{$sum_of_mismatches}->{$alignment_location}->{chromosome}=$chromosome_1; # either is fine
|
|
1886 $mismatches{$sum_of_mismatches}->{$alignment_location}->{start_seq_1}=$position_1;
|
|
1887 $mismatches{$sum_of_mismatches}->{$alignment_location}->{start_seq_2}=$position_2;
|
|
1888 $mismatches{$sum_of_mismatches}->{$alignment_location}->{number_of_mismatches_1} = $number_of_mismatches_1;
|
|
1889 $mismatches{$sum_of_mismatches}->{$alignment_location}->{number_of_mismatches_2} = $number_of_mismatches_2;
|
|
1890 }
|
|
1891 ###################################################################################################################################################
|
|
1892 ### STEP II Now reading in the next 2 lines from the bowtie filehandle. If there are 2 next lines in the alignments filehandle it can either ###
|
|
1893 ### be a second alignment of the same sequence pair or a new sequence pair. In any case we will just add it to last_line_1 and last_line _2. ###
|
|
1894 ### If it is the alignment of the next sequence pair, 0 will be returned as $valid_alignment_found, so it will not be processed any further in ###
|
|
1895 ### this round ###
|
|
1896 ###################################################################################################################################################
|
|
1897 my $newline_1 = $fhs[$index]->{fh}-> getline();
|
|
1898 my $newline_2 = $fhs[$index]->{fh}-> getline();
|
|
1899
|
|
1900 if ($newline_1 and $newline_2){
|
|
1901 my ($seq_id_1) = split (/\t/,$newline_1);
|
|
1902 my ($seq_id_2) = split (/\t/,$newline_2);
|
|
1903
|
|
1904 if ($seq_id_1 =~ s/\/1$//){ # removing the read /1 tag
|
|
1905 $fhs[$index]->{last_seq_id} = $seq_id_1;
|
|
1906 }
|
|
1907 elsif ($seq_id_2 =~ s/\/1$//){ # removing the read /1 tag
|
|
1908 $fhs[$index]->{last_seq_id} = $seq_id_2;
|
|
1909 }
|
|
1910 else{
|
|
1911 die "Either read 1 or read 2 needs to end on '/1'\n";
|
|
1912 }
|
|
1913
|
|
1914 $fhs[$index]->{last_line_1} = $newline_1;
|
|
1915 $fhs[$index]->{last_line_2} = $newline_2;
|
|
1916 }
|
|
1917 else {
|
|
1918 # assigning undef to last_seq_id and both last_lines and jumping to the next index (end of bowtie output)
|
|
1919 $fhs[$index]->{last_seq_id} = undef;
|
|
1920 $fhs[$index]->{last_line_1} = undef;
|
|
1921 $fhs[$index]->{last_line_2} = undef;
|
|
1922 next; # jumping to the next index
|
|
1923 }
|
|
1924 ### Now processing the entry we just stored in last_line_1 and last_line_2
|
|
1925 $valid_alignment_found = decide_whether_paired_end_alignment_is_valid($index,$identifier);
|
|
1926 ### only processing the alignment further if 1 was returned. 0 will be returned either if the alignment is already the next sequence pair to
|
|
1927 ### be analysed or if it was a second alignment of the current sequence pair but in the wrong orientation
|
|
1928 if ($valid_alignment_found == 1){
|
|
1929 ### we store the useful information in %mismatches
|
|
1930 ($id_1,$strand_1,$mapped_chromosome_1,$position_1,$bowtie_sequence_1,$mismatch_info_1) = (split (/\t/,$fhs[$index]->{last_line_1}))[0,1,2,3,4,7];
|
|
1931 ($id_2,$strand_2,$mapped_chromosome_2,$position_2,$bowtie_sequence_2,$mismatch_info_2) = (split (/\t/,$fhs[$index]->{last_line_2}))[0,1,2,3,4,7];
|
|
1932 chomp $mismatch_info_1;
|
|
1933 chomp $mismatch_info_2;
|
|
1934 ### need to extract the chromosome number from the bowtie output (which is either _CT_converted or _GA_converted)
|
|
1935 if ($mapped_chromosome_1 =~ s/_(CT|GA)_converted$//){
|
|
1936 $chromosome_1 = $mapped_chromosome_1;
|
|
1937 }
|
|
1938 else{
|
|
1939 die "Chromosome number extraction failed for $mapped_chromosome_1\n";
|
|
1940 }
|
|
1941 if ($mapped_chromosome_2 =~ s/_(CT|GA)_converted$//){
|
|
1942 $chromosome_2 = $mapped_chromosome_2;
|
|
1943 }
|
|
1944 else{
|
|
1945 die "Chromosome number extraction failed for $mapped_chromosome_2\n";
|
|
1946 }
|
|
1947
|
|
1948 $number_of_mismatches_1='';
|
|
1949 $number_of_mismatches_2='';
|
|
1950 ### Now extracting the number of mismatches to the converted genome
|
|
1951 if ($mismatch_info_1 eq ''){
|
|
1952 $number_of_mismatches_1 = 0;
|
|
1953 }
|
|
1954 elsif ($mismatch_info_1 =~ /^\d/){
|
|
1955 my @mismatches = split (/,/,$mismatch_info_1);
|
|
1956 $number_of_mismatches_1 = scalar @mismatches;
|
|
1957 }
|
|
1958 else{
|
|
1959 die "Something weird is going on with the mismatch field\n";
|
|
1960 }
|
|
1961 if ($mismatch_info_2 eq ''){
|
|
1962 $number_of_mismatches_2 = 0;
|
|
1963 }
|
|
1964 elsif ($mismatch_info_2 =~ /^\d/){
|
|
1965 my @mismatches = split (/,/,$mismatch_info_2);
|
|
1966 $number_of_mismatches_2 = scalar @mismatches;
|
|
1967 }
|
|
1968 else{
|
|
1969 die "Something weird is going on with the mismatch field\n";
|
|
1970 }
|
|
1971 ### To decide whether a sequence pair has a unique best alignment we will look at the lowest sum of mismatches from both alignments
|
|
1972 $sum_of_mismatches = $number_of_mismatches_1+$number_of_mismatches_2;
|
|
1973 ### creating a composite location variable from $chromosome and $position and storing the alignment information in a temporary hash table
|
|
1974 die "position 1 is greater than position 2" if ($position_1 > $position_2);
|
|
1975 die "Paired-end alignments need to be on the same chromosome\n" unless ($chromosome_1 eq $chromosome_2);
|
|
1976 $alignment_location = join(":",$chromosome_1,$position_1,$position_2);
|
|
1977 ### If a sequence aligns to exactly the same location twice the sequence does either not contain any C or G, or all the Cs (or Gs on the reverse
|
|
1978 ### strand) were methylated and therefore protected. It is not needed to overwrite the same positional entry with a second entry for the same
|
|
1979 ### location (the genomic sequence extraction and methylation would not be affected by this, only the thing which would change is the index
|
|
1980 ### number for the found alignment)
|
|
1981 unless (exists $mismatches{$sum_of_mismatches}->{$alignment_location}){
|
|
1982 $mismatches{$sum_of_mismatches}->{$alignment_location}->{seq_id}=$id_1; # either is fine
|
|
1983 $mismatches{$sum_of_mismatches}->{$alignment_location}->{bowtie_sequence_1}=$bowtie_sequence_1;
|
|
1984 $mismatches{$sum_of_mismatches}->{$alignment_location}->{bowtie_sequence_2}=$bowtie_sequence_2;
|
|
1985 $mismatches{$sum_of_mismatches}->{$alignment_location}->{index}=$index;
|
|
1986 $mismatches{$sum_of_mismatches}->{$alignment_location}->{chromosome}=$chromosome_1; # either is fine
|
|
1987 $mismatches{$sum_of_mismatches}->{$alignment_location}->{start_seq_1}=$position_1;
|
|
1988 $mismatches{$sum_of_mismatches}->{$alignment_location}->{start_seq_2}=$position_2;
|
|
1989 $mismatches{$sum_of_mismatches}->{$alignment_location}->{number_of_mismatches_1} = $number_of_mismatches_1;
|
|
1990 $mismatches{$sum_of_mismatches}->{$alignment_location}->{number_of_mismatches_2} = $number_of_mismatches_2;
|
|
1991 }
|
|
1992 ###############################################################################################################################################
|
|
1993 ### STEP III Now reading in two more lines. These have to be the next entry and we will just add assign them to last_line_1 and last_line_2 ###
|
|
1994 ###############################################################################################################################################
|
|
1995 $newline_1 = $fhs[$index]->{fh}-> getline();
|
|
1996 $newline_2 = $fhs[$index]->{fh}-> getline();
|
|
1997
|
|
1998 if ($newline_1 and $newline_2){
|
|
1999 my ($seq_id_1) = split (/\t/,$newline_1);
|
|
2000 my ($seq_id_2) = split (/\t/,$newline_2);
|
|
2001
|
|
2002 if ($seq_id_1 =~ s/\/1$//){ # removing the read /1 tag
|
|
2003 $fhs[$index]->{last_seq_id} = $seq_id_1;
|
|
2004 }
|
|
2005 if ($seq_id_2 =~ s/\/1$//){ # removing the read /1 tag
|
|
2006 $fhs[$index]->{last_seq_id} = $seq_id_2;
|
|
2007 }
|
|
2008 $fhs[$index]->{last_line_1} = $newline_1;
|
|
2009 $fhs[$index]->{last_line_2} = $newline_2;
|
|
2010 }
|
|
2011 else {
|
|
2012 # assigning undef to last_seq_id and both last_lines and jumping to the next index (end of bowtie output)
|
|
2013 $fhs[$index]->{last_seq_id} = undef;
|
|
2014 $fhs[$index]->{last_line_1} = undef;
|
|
2015 $fhs[$index]->{last_line_2} = undef;
|
|
2016 next; # jumping to the next index
|
|
2017 }
|
|
2018 ### within the 2nd sequence pair alignment in correct orientation found
|
|
2019 }
|
|
2020 ### within the 1st sequence pair alignment in correct orientation found
|
|
2021 }
|
|
2022 ### still within the (last_seq_id eq identifier) condition
|
|
2023 }
|
|
2024 ### still within foreach index loop
|
|
2025 }
|
|
2026 ### if there was no single alignment found for a certain sequence we will continue with the next sequence in the sequence file
|
|
2027 unless(%mismatches){
|
|
2028 $counting{no_single_alignment_found}++;
|
|
2029 return 1; ### We will print this sequence out as unmapped sequence if --un unmapped.out has been specified
|
|
2030 }
|
|
2031 ### Going to use the variable $sequence_pair_fails as a 'memory' if a sequence could not be aligned uniquely (set to 1 then)
|
|
2032 my $sequence_pair_fails = 0;
|
|
2033 ### Declaring an empty hash reference which will store all information we need for the methylation call
|
|
2034 my $methylation_call_params; # hash reference!
|
|
2035 ### We are now looking if there is a unique best alignment for a certain sequence. This means we are sorting in ascending order and look at the
|
|
2036 ### sequence with the lowest amount of mismatches. If there is only one single best position we are going to store the alignment information in the
|
|
2037 ### meth_call variables, if there are multiple hits with the same amount of (lowest) mismatches we are discarding the sequence altogether
|
|
2038 foreach my $mismatch_number (sort {$a<=>$b} keys %mismatches){
|
|
2039 #dev print "Number of mismatches: $mismatch_number\t$identifier\t$sequence_1\t$sequence_2\n";
|
|
2040 foreach my $entry (keys (%{$mismatches{$mismatch_number}}) ){
|
|
2041 #dev print "$mismatch_number\t$entry\t$mismatches{$mismatch_number}->{$entry}->{index}\n";
|
|
2042 # print join("\t",$mismatch_number,$mismatches{$mismatch_number}->{$entry}->{seq_id},$sequence,$mismatches{$mismatch_number}->{$entry}->{bowtie_sequence},$mismatches{$mismatch_number}->{$entry}->{chromosome},$mismatches{$mismatch_number}->{$entry}->{position},$mismatches{$mismatch_number}->{$entry}->{index}),"\n";
|
|
2043 }
|
|
2044 if (scalar keys %{$mismatches{$mismatch_number}} == 1){
|
|
2045 # print "Unique best alignment for sequence pair $sequence_1\t$sequence_1\n";
|
|
2046 for my $unique_best_alignment (keys %{$mismatches{$mismatch_number}}){
|
|
2047 $methylation_call_params->{$identifier}->{seq_id} = $identifier;
|
|
2048 $methylation_call_params->{$identifier}->{bowtie_sequence_1} = $mismatches{$mismatch_number}->{$unique_best_alignment}->{bowtie_sequence_1};
|
|
2049 $methylation_call_params->{$identifier}->{bowtie_sequence_2} = $mismatches{$mismatch_number}->{$unique_best_alignment}->{bowtie_sequence_2};
|
|
2050 $methylation_call_params->{$identifier}->{chromosome} = $mismatches{$mismatch_number}->{$unique_best_alignment}->{chromosome};
|
|
2051 $methylation_call_params->{$identifier}->{start_seq_1} = $mismatches{$mismatch_number}->{$unique_best_alignment}->{start_seq_1};
|
|
2052 $methylation_call_params->{$identifier}->{start_seq_2} = $mismatches{$mismatch_number}->{$unique_best_alignment}->{start_seq_2};
|
|
2053 $methylation_call_params->{$identifier}->{alignment_end} = ($mismatches{$mismatch_number}->{$unique_best_alignment}->{start_seq_2}+length($mismatches{$mismatch_number}->{$unique_best_alignment}->{bowtie_sequence_2}));
|
|
2054 $methylation_call_params->{$identifier}->{index} = $mismatches{$mismatch_number}->{$unique_best_alignment}->{index};
|
|
2055 $methylation_call_params->{$identifier}->{number_of_mismatches_1} = $mismatches{$mismatch_number}->{$unique_best_alignment}->{number_of_mismatches_1};
|
|
2056 $methylation_call_params->{$identifier}->{number_of_mismatches_2} = $mismatches{$mismatch_number}->{$unique_best_alignment}->{number_of_mismatches_2};
|
|
2057 }
|
|
2058 }
|
|
2059 else{
|
|
2060 $sequence_pair_fails = 1;
|
|
2061 }
|
|
2062 ### after processing the alignment with the lowest number of mismatches we exit
|
|
2063 last;
|
|
2064 }
|
|
2065 ### skipping the sequence completely if there were multiple alignments with the same amount of lowest mismatches found at different positions
|
|
2066 if ($sequence_pair_fails == 1){
|
|
2067 $counting{unsuitable_sequence_count}++;
|
|
2068 if ($ambiguous){
|
|
2069 return 2; # => exits to next sequence pair, and prints both seqs out to multiple_alignments_1 and -2 if --ambiguous has been specified
|
|
2070 }
|
|
2071 if ($unmapped){
|
|
2072 return 1; # => exits to next sequence pair, and prints both seqs out to unmapped_1 and _2 if --un has been specified
|
|
2073 }
|
|
2074 else{
|
|
2075 return 0; # => exits to next sequence (default)
|
|
2076 }
|
|
2077 }
|
|
2078
|
|
2079 ### --DIRECTIONAL
|
|
2080 ### If the option --directional has been specified the user wants to consider only alignments to the original top strand or the original bottom strand. We will therefore
|
|
2081 ### discard all alignments to strands complementary to the original strands, as they should not exist in reality due to the library preparation protocol
|
|
2082 if ($directional){
|
|
2083 if ( ($methylation_call_params->{$identifier}->{index} == 1) or ($methylation_call_params->{$identifier}->{index} == 2) ){
|
|
2084 # warn "Alignment rejected! (index was: $methylation_call_params->{$identifier}->{index})\n";
|
|
2085 $counting{alignments_rejected_count}++;
|
|
2086 return 0;
|
|
2087 }
|
|
2088 }
|
|
2089
|
|
2090 ### If the sequence has not been rejected so far it does have a unique best alignment
|
|
2091 $counting{unique_best_alignment_count}++;
|
|
2092 extract_corresponding_genomic_sequence_paired_ends($identifier,$methylation_call_params);
|
|
2093
|
|
2094 ### check test to see if the genomic sequences we extracted has the same length as the observed sequences +2, and only then we perform the methylation call
|
|
2095 if (length($methylation_call_params->{$identifier}->{unmodified_genomic_sequence_1}) != length($sequence_1)+2){
|
|
2096 warn "Chromosomal sequence could not be extracted for\t$identifier\t$methylation_call_params->{$identifier}->{chromosome}\t$methylation_call_params->{$identifier}->{start_seq_1}\n";
|
|
2097 $counting{genomic_sequence_could_not_be_extracted_count}++;
|
|
2098 return 0;
|
|
2099 }
|
|
2100 if (length($methylation_call_params->{$identifier}->{unmodified_genomic_sequence_2}) != length($sequence_2)+2){
|
|
2101 warn "Chromosomal sequence could not be extracted for\t$identifier\t$methylation_call_params->{$identifier}->{chromosome}\t$methylation_call_params->{$identifier}->{start_seq_2}\n";
|
|
2102 $counting{genomic_sequence_could_not_be_extracted_count}++;
|
|
2103 return 0;
|
|
2104 }
|
|
2105
|
|
2106 ### otherwise we are set to perform the actual methylation call
|
|
2107 $methylation_call_params->{$identifier}->{methylation_call_1} = methylation_call($identifier,$sequence_1,$methylation_call_params->{$identifier}->{unmodified_genomic_sequence_1},$methylation_call_params->{$identifier}->{read_conversion_1});
|
|
2108 $methylation_call_params->{$identifier}->{methylation_call_2} = methylation_call($identifier,$sequence_2,$methylation_call_params->{$identifier}->{unmodified_genomic_sequence_2},$methylation_call_params->{$identifier}->{read_conversion_2});
|
|
2109
|
|
2110 print_bisulfite_mapping_results_paired_ends($identifier,$sequence_1,$sequence_2,$methylation_call_params,$quality_value_1,$quality_value_2);
|
|
2111 return 0; ## otherwise 1 will be returned by default, which would print the sequence pair to unmapped_1 and _2
|
|
2112 }
|
|
2113
|
|
2114 #########################
|
|
2115 ### BOWTIE 2 | PAIRED-END
|
|
2116 #########################
|
|
2117
|
|
2118 sub check_bowtie_results_paired_ends_bowtie2{
|
|
2119 my ($sequence_1,$sequence_2,$identifier,$quality_value_1,$quality_value_2) = @_;
|
|
2120
|
|
2121 ### quality values are not given for FastA files, so they are initialised with a Phred quality of 40
|
|
2122 unless ($quality_value_1){
|
|
2123 $quality_value_1 = 'I'x(length$sequence_1);
|
|
2124 }
|
|
2125
|
|
2126 unless ($quality_value_2){
|
|
2127 $quality_value_2 = 'I'x(length$sequence_2);
|
|
2128 }
|
|
2129
|
|
2130
|
|
2131 # print "$identifier\n$fhs[0]->{last_seq_id}\n$fhs[1]->{last_seq_id}\n$fhs[2]->{last_seq_id}\n$fhs[3]->{last_seq_id}\n\n";
|
|
2132
|
|
2133
|
|
2134 my %alignments;
|
|
2135 my $alignment_ambiguous = 0;
|
|
2136
|
|
2137 ### reading from the Bowtie 2 output filehandles
|
|
2138
|
|
2139 ### for paired end reads we are reporting alignments to the OT strand first (index 0), then the OB strand (index 3!!), similiar to the single end way.
|
|
2140 ### alignments to the complementary strands are reported afterwards (CTOT got index 1, and CTOB got index 2).
|
|
2141 ### This is needed so that alignments which either contain no single C or G or reads which contain only protected Cs are reported to the original strands (OT and OB)
|
|
2142 ### Before the complementary strands. Remember that it does not make any difference for the methylation calls, but it will matter if alignments to the complementary
|
|
2143 ### strands are not being reported when '--directional' is specified
|
|
2144
|
|
2145 foreach my $index (0,3,1,2){
|
|
2146 ### skipping this index if the last alignment has been set to undefined already (i.e. end of bowtie output)
|
|
2147 next unless ($fhs[$index]->{last_line_1} and $fhs[$index]->{last_line_2} and defined $fhs[$index]->{last_seq_id});
|
|
2148
|
|
2149 ### if the sequence pair we are currently looking at produced an alignment we are doing various things with it
|
|
2150 if ($fhs[$index]->{last_seq_id} eq $identifier) {
|
|
2151
|
|
2152 my ($id_1,$flag_1,$mapped_chromosome_1,$position_1,$mapping_quality_1,$cigar_1,$bowtie_sequence_1,$qual_1) = (split (/\t/,$fhs[$index]->{last_line_1}))[0,1,2,3,4,5,9,10];
|
|
2153 my ($id_2,$flag_2,$mapped_chromosome_2,$position_2,$mapping_quality_2,$cigar_2,$bowtie_sequence_2,$qual_2) = (split (/\t/,$fhs[$index]->{last_line_2}))[0,1,2,3,4,5,9,10];
|
|
2154 # print "Index: $index\t$fhs[$index]->{last_line_1}\n";
|
|
2155 # print "Index: $index\t$fhs[$index]->{last_line_2}\n";
|
|
2156 # print join ("\t",$id_1,$flag_1,$mapped_chromosome_1,$position_1,$mapping_quality_1,$cigar_1,$bowtie_sequence_1,$qual_1),"\n";
|
|
2157 # print join ("\t",$id_2,$flag_2,$mapped_chromosome_2,$position_2,$mapping_quality_2,$cigar_2,$bowtie_sequence_2,$qual_2),"\n";
|
|
2158 $id_1 =~ s/\/1$//;
|
|
2159 $id_2 =~ s/\/2$//;
|
|
2160
|
|
2161 # SAM format specifications for Bowtie 2
|
|
2162 # (1) Name of read that aligned
|
|
2163 # (2) Sum of all applicable flags. Flags relevant to Bowtie are:
|
|
2164 # 1 The read is one of a pair
|
|
2165 # 2 The alignment is one end of a proper paired-end alignment
|
|
2166 # 4 The read has no reported alignments
|
|
2167 # 8 The read is one of a pair and has no reported alignments
|
|
2168 # 16 The alignment is to the reverse reference strand
|
|
2169 # 32 The other mate in the paired-end alignment is aligned to the reverse reference strand
|
|
2170 # 64 The read is mate 1 in a pair
|
|
2171 # 128 The read is mate 2 in a pair
|
|
2172 # 256 The read has multiple mapping states
|
|
2173 # (3) Name of reference sequence where alignment occurs (unmapped reads have a *)
|
|
2174 # (4) 1-based offset into the forward reference strand where leftmost character of the alignment occurs (0 for unmapped reads)
|
|
2175 # (5) Mapping quality (255 means MAPQ is not available)
|
|
2176 # (6) CIGAR string representation of alignment (* if unavailable)
|
|
2177 # (7) Name of reference sequence where mate's alignment occurs. Set to = if the mate's reference sequence is the same as this alignment's, or * if there is no mate.
|
|
2178 # (8) 1-based offset into the forward reference strand where leftmost character of the mate's alignment occurs. Offset is 0 if there is no mate.
|
|
2179 # (9) Inferred fragment size. Size is negative if the mate's alignment occurs upstream of this alignment. Size is 0 if there is no mate.
|
|
2180 # (10) Read sequence (reverse-complemented if aligned to the reverse strand)
|
|
2181 # (11) ASCII-encoded read qualities (reverse-complemented if the read aligned to the reverse strand). The encoded quality values are on the Phred quality scale and the encoding is ASCII-offset by 33 (ASCII char !), similarly to a FASTQ file.
|
|
2182 # (12) Optional fields. Fields are tab-separated. bowtie2 outputs zero or more of these optional fields for each alignment, depending on the type of the alignment:
|
|
2183 # AS:i:<N> Alignment score. Can be negative. Can be greater than 0 in --local mode (but not in --end-to-end mode). Only present if SAM record is for an aligned read.
|
|
2184 # XS:i:<N> Alignment score for second-best alignment. Can be negative. Can be greater than 0 in --local mode (but not in --end-to-end mode). Only present if the SAM record is for an aligned read and more than one alignment was found for the read.
|
|
2185 # YS:i:<N> Alignment score for opposite mate in the paired-end alignment. Only present if the SAM record is for a read that aligned as part of a paired-end alignment.
|
|
2186 # XN:i:<N> The number of ambiguous bases in the reference covering this alignment. Only present if SAM record is for an aligned read.
|
|
2187 # XM:i:<N> The number of mismatches in the alignment. Only present if SAM record is for an aligned read.
|
|
2188 # XO:i:<N> The number of gap opens, for both read and reference gaps, in the alignment. Only present if SAM record is for an aligned read.
|
|
2189 # XG:i:<N> The number of gap extensions, for both read and reference gaps, in the alignment. Only present if SAM record is for an aligned read.
|
|
2190 # NM:i:<N> The edit distance; that is, the minimal number of one-nucleotide edits (substitutions, insertions and deletions) needed to transform the read string into the reference string. Only present if SAM record is for an aligned read.
|
|
2191 # YF:Z:<N> String indicating reason why the read was filtered out. See also: Filtering. Only appears for reads that were filtered out.
|
|
2192 # MD:Z:<S> A string representation of the mismatched reference bases in the alignment. See SAM format specification for details. Only present if SAM record is for an aligned read.
|
|
2193
|
|
2194 ### If a sequence has no reported alignments there will be a single output line per sequence with a bit-wise flag value of 77 for read 1 (1+4+8+64), or 141 for read 2 (1+4+8+128).
|
|
2195 ### We can store the next alignment and move on to the next Bowtie 2 instance
|
|
2196 if ($flag_1 == 77 and $flag_2 == 141){
|
|
2197 ## reading in the next alignment, which must be the next sequence
|
|
2198 my $newline_1 = $fhs[$index]->{fh}-> getline();
|
|
2199 my $newline_2 = $fhs[$index]->{fh}-> getline();
|
|
2200
|
|
2201 if ($newline_1 and $newline_2){
|
|
2202 chomp $newline_1;
|
|
2203 chomp $newline_2;
|
|
2204 my ($seq_id_1) = split (/\t/,$newline_1);
|
|
2205 my ($seq_id_2) = split (/\t/,$newline_2);
|
|
2206 $seq_id_1 =~ s/\/1$//;
|
|
2207 $seq_id_2 =~ s/\/2$//;
|
|
2208 $fhs[$index]->{last_seq_id} = $seq_id_1;
|
|
2209 $fhs[$index]->{last_line_1} = $newline_1;
|
|
2210 $fhs[$index]->{last_line_2} = $newline_2;
|
|
2211
|
|
2212 # print "current sequence ($identifier) did not map, reading in next sequence\n";
|
|
2213 # print "$index\t$fhs[$index]->{last_seq_id}\n";
|
|
2214 # print "$index\t$fhs[$index]->{last_line_1}\n";
|
|
2215 # print "$index\t$fhs[$index]->{last_line_2}\n";
|
|
2216 next; # next instance
|
|
2217 }
|
|
2218 else{
|
|
2219 # assigning undef to last_seq_id and last_line and jumping to the next index (end of Bowtie 2 output)
|
|
2220 $fhs[$index]->{last_seq_id} = undef;
|
|
2221 $fhs[$index]->{last_line_1} = undef;
|
|
2222 $fhs[$index]->{last_line_2} = undef;
|
|
2223 next;
|
|
2224 }
|
|
2225 }
|
|
2226
|
|
2227 ### If there are one or more proper alignments we can extract the chromosome number
|
|
2228 my ($chromosome_1,$chromosome_2);
|
|
2229 if ($mapped_chromosome_1 =~ s/_(CT|GA)_converted$//){
|
|
2230 $chromosome_1 = $mapped_chromosome_1;
|
|
2231 }
|
|
2232 else{
|
|
2233 die "Chromosome number extraction failed for $mapped_chromosome_1\n";
|
|
2234 }
|
|
2235 if ($mapped_chromosome_2 =~ s/_(CT|GA)_converted$//){
|
|
2236 $chromosome_2 = $mapped_chromosome_2;
|
|
2237 }
|
|
2238 else{
|
|
2239 die "Chromosome number extraction failed for $mapped_chromosome_2\n";
|
|
2240 }
|
|
2241
|
|
2242 die "Paired-end alignments need to be on the same chromosome\n" unless ($chromosome_1 eq $chromosome_2);
|
|
2243
|
|
2244 ### We will use the optional fields to determine the best alignments. Later on we extract the number of mismatches and/or indels from the CIGAR string
|
|
2245 my ($alignment_score_1,$alignment_score_2,$second_best_1,$second_best_2,$MD_tag_1,$MD_tag_2);
|
|
2246
|
|
2247 my @fields_1 = split (/\t/,$fhs[$index]->{last_line_1});
|
|
2248 my @fields_2 = split (/\t/,$fhs[$index]->{last_line_2});
|
|
2249
|
|
2250 foreach (11..$#fields_1){
|
|
2251 if ($fields_1[$_] =~ /AS:i:(.*)/){
|
|
2252 $alignment_score_1 = $1;
|
|
2253 }
|
|
2254 elsif ($fields_1[$_] =~ /XS:i:(.*)/){
|
|
2255 $second_best_1 = $1;
|
|
2256 }
|
|
2257 elsif ($fields_1[$_] =~ /MD:Z:(.*)/){
|
|
2258 $MD_tag_1 = $1;
|
|
2259 }
|
|
2260 }
|
|
2261
|
|
2262 foreach (11..$#fields_2){
|
|
2263 if ($fields_2[$_] =~ /AS:i:(.*)/){
|
|
2264 $alignment_score_2 = $1;
|
|
2265 }
|
|
2266 elsif ($fields_2[$_] =~ /XS:i:(.*)/){
|
|
2267 $second_best_2 = $1;
|
|
2268 }
|
|
2269 elsif ($fields_2[$_] =~ /MD:Z:(.*)/){
|
|
2270 $MD_tag_2 = $1;
|
|
2271 }
|
|
2272 }
|
|
2273
|
|
2274 die "Failed to extract alignment score 1 ($alignment_score_1) and MD tag ($MD_tag_1)!\nlast alignment 1: $fhs[$index]->{last_line_1}\nlast alignment 2: $fhs[$index]->{last_line_2}\n" unless (defined $alignment_score_1 and defined $MD_tag_1);
|
|
2275 die "Failed to extract alignment score 2 ($alignment_score_2) and MD tag ($MD_tag_2)!\nlast alignment 1: $fhs[$index]->{last_line_1}\nlast alignment 2: $fhs[$index]->{last_line_2}\n" unless (defined $alignment_score_2 and defined $MD_tag_2);
|
|
2276
|
|
2277 # warn "First read 1 alignment score is: '$alignment_score_1'\n";
|
|
2278 # warn "First read 2 alignment score is: '$alignment_score_2'\n";
|
|
2279 # warn "MD tag 1 is: '$MD_tag_1'\n";
|
|
2280 # warn "MD tag 2 is: '$MD_tag_2'\n";
|
|
2281
|
|
2282 ### To decide whether a sequence pair has a unique best alignment we will look at the highest sum of alignment scores from both alignments
|
|
2283 my $sum_of_alignment_scores_1 = $alignment_score_1 + $alignment_score_2 ;
|
|
2284 # print "sum of alignment scores: $sum_of_alignment_scores_1\n\n";
|
|
2285
|
|
2286 if (defined $second_best_1 and defined $second_best_2){
|
|
2287 my $sum_of_alignment_scores_second_best = $second_best_1 + $second_best_2;
|
|
2288 # warn "Second best alignment_score_1 is: '$second_best_1'\n";
|
|
2289 # warn "Second best alignment_score_2 is: '$second_best_2'\n";
|
|
2290 # warn "Second best alignment sum of alignment scores is: '$sum_of_alignment_scores_second_best'\n";
|
|
2291
|
|
2292 # If the first alignment score for the first read pair is the same as the alignment score of the second best hit we are going to boot this sequence pair altogether
|
|
2293 if ($sum_of_alignment_scores_1 == $sum_of_alignment_scores_second_best){
|
|
2294 $alignment_ambiguous = 1;
|
|
2295 # print "This read will be chucked (AS==XS detected)!\n";
|
|
2296
|
|
2297 ## need to read and discard all additional ambiguous reads until we reach the next sequence
|
|
2298 until ($fhs[$index]->{last_seq_id} ne $identifier){
|
|
2299 my $newline_1 = $fhs[$index]->{fh}-> getline();
|
|
2300 my $newline_2 = $fhs[$index]->{fh}-> getline();
|
|
2301 if ($newline_1 and $newline_2){
|
|
2302 chomp $newline_1;
|
|
2303 chomp $newline_2;
|
|
2304 my ($seq_id_1) = split (/\t/,$newline_1);
|
|
2305 my ($seq_id_2) = split (/\t/,$newline_2);
|
|
2306 $seq_id_1 =~ s/\/1$//;
|
|
2307 $seq_id_2 =~ s/\/2$//;
|
|
2308 # print "New Seq IDs:\t$seq_id_1\t$seq_id_2\n";
|
|
2309
|
|
2310 $fhs[$index]->{last_seq_id} = $seq_id_1;
|
|
2311 $fhs[$index]->{last_line_1} = $newline_1;
|
|
2312 $fhs[$index]->{last_line_2} = $newline_2;
|
|
2313 }
|
|
2314 else{
|
|
2315 # assigning undef to last_seq_id and last_line and jumping to the next index (end of Bowtie 2 output)
|
|
2316 $fhs[$index]->{last_seq_id} = undef;
|
|
2317 $fhs[$index]->{last_line_1} = undef;
|
|
2318 $fhs[$index]->{last_line_2} = undef;
|
|
2319 last; # break free if the end of the alignment output was reached
|
|
2320 }
|
|
2321 }
|
|
2322 # if ($fhs[$index]->{last_seq_id}){
|
|
2323 # warn "Index: $index\tThis Seq-ID is $identifier, skipped all ambiguous sequences until the next ID which is: $fhs[$index]->{last_seq_id}\n";
|
|
2324 # }
|
|
2325 }
|
|
2326 else{ # the next best alignment has a lower alignment score than the current read, so we can safely store the current alignment
|
|
2327
|
|
2328 my $alignment_location;
|
|
2329 if ($position_1 <= $position_2){
|
|
2330 $alignment_location = join(":",$chromosome_1,$position_1,$position_2);
|
|
2331 }
|
|
2332 elsif($position_2 < $position_1){
|
|
2333 $alignment_location = join(":",$chromosome_1,$position_2,$position_1);
|
|
2334 }
|
|
2335
|
|
2336 ### If a sequence aligns to exactly the same location twice the sequence does either not contain any C or G, or all the Cs (or Gs on the reverse
|
|
2337 ### strand) were methylated and therefore protected. Alternatively it will align better in one condition than in the other. In any case, it is not needed to overwrite
|
|
2338 ### the same positional entry with a second entry for the same location, as the genomic sequence extraction and methylation call would not be affected by this. The only
|
|
2339 ### thing which would change is the index number for the found alignment). We will continue to assign these alignments to the first indexes 0 and 3, i.e. OT and OB
|
|
2340
|
|
2341 unless (exists $alignments{$alignment_location}){
|
|
2342 $alignments{$alignment_location}->{seq_id} = $id_1;
|
|
2343 $alignments{$alignment_location}->{alignment_score_1} = $alignment_score_1;
|
|
2344 $alignments{$alignment_location}->{alignment_score_2} = $alignment_score_2;
|
|
2345 $alignments{$alignment_location}->{sum_of_alignment_scores} = $sum_of_alignment_scores_1;
|
|
2346 $alignments{$alignment_location}->{bowtie_sequence_1} = $bowtie_sequence_1;
|
|
2347 $alignments{$alignment_location}->{bowtie_sequence_2} = $bowtie_sequence_2;
|
|
2348 $alignments{$alignment_location}->{index} = $index;
|
|
2349 $alignments{$alignment_location}->{chromosome} = $chromosome_1; # either is fine
|
|
2350 $alignments{$alignment_location}->{position_1} = $position_1;
|
|
2351 $alignments{$alignment_location}->{position_2} = $position_2;
|
|
2352 $alignments{$alignment_location}->{mismatch_info_1} = $MD_tag_1;
|
|
2353 $alignments{$alignment_location}->{mismatch_info_2} = $MD_tag_2;
|
|
2354 $alignments{$alignment_location}->{CIGAR_1} = $cigar_1;
|
|
2355 $alignments{$alignment_location}->{CIGAR_2} = $cigar_2;
|
|
2356 $alignments{$alignment_location}->{flag_1} = $flag_1;
|
|
2357 $alignments{$alignment_location}->{flag_2} = $flag_2;
|
|
2358 }
|
|
2359 # warn "added best of several alignments to \%alignments hash\n";
|
|
2360
|
|
2361 ### now reading and discarding all (inferior) alignments of this read pair until we hit the next sequence
|
|
2362 until ($fhs[$index]->{last_seq_id} ne $identifier){
|
|
2363 my $newline_1 = $fhs[$index]->{fh}-> getline();
|
|
2364 my $newline_2 = $fhs[$index]->{fh}-> getline();
|
|
2365 if ($newline_1 and $newline_2){
|
|
2366 chomp $newline_1;
|
|
2367 chomp $newline_2;
|
|
2368 my ($seq_id_1) = split (/\t/,$newline_1);
|
|
2369 my ($seq_id_2) = split (/\t/,$newline_2);
|
|
2370 $seq_id_1 =~ s/\/1$//;
|
|
2371 $seq_id_2 =~ s/\/2$//;
|
|
2372 # print "New Seq IDs:\t$seq_id_1\t$seq_id_2\n";
|
|
2373
|
|
2374 $fhs[$index]->{last_seq_id} = $seq_id_1;
|
|
2375 $fhs[$index]->{last_line_1} = $newline_1;
|
|
2376 $fhs[$index]->{last_line_2} = $newline_2;
|
|
2377 }
|
|
2378 else{
|
|
2379 # assigning undef to last_seq_id and last_line_1 and _2 and jumping to the next index (end of Bowtie 2 output)
|
|
2380 $fhs[$index]->{last_seq_id} = undef;
|
|
2381 $fhs[$index]->{last_line_1} = undef;
|
|
2382 $fhs[$index]->{last_line_2} = undef;
|
|
2383 last; # break free if the end of the alignment output was reached
|
|
2384 }
|
|
2385 }
|
|
2386 # if($fhs[$index]->{last_seq_id}){
|
|
2387 # warn "Index: $index\tThis Seq-ID is $identifier, skipped all other alignments until the next ID was reached which is: $fhs[$index]->{last_seq_id}\n";
|
|
2388 # }
|
|
2389 }
|
|
2390 }
|
|
2391 else{ # there is no second best hit, so we can just store this one and read in the next sequence
|
|
2392
|
|
2393 my $alignment_location = join(":",$chromosome_1,$position_1,$position_2);
|
|
2394 # print "$alignment_location\n";
|
|
2395 ### If a sequence aligns to exactly the same location with a perfect match twice the sequence does either not contain any C or G, or all the Cs (or Gs on the reverse
|
|
2396 ### strand) were methylated and therefore protected. Alternatively it will align better in one condition than in the other. In any case, it is not needed to overwrite
|
|
2397 ### the same positional entry with a second entry for the same location, as the genomic sequence extraction and methylation call would not be affected by this. The only
|
|
2398 ### thing which would change is the index number for the found alignment). We will continue to assign these alignments to the first indexes 0 and 3, i.e. OT and OB
|
|
2399
|
|
2400 unless (exists $alignments{$alignment_location}){
|
|
2401 $alignments{$alignment_location}->{seq_id} = $id_1;
|
|
2402 $alignments{$alignment_location}->{alignment_score_1} = $alignment_score_1;
|
|
2403 $alignments{$alignment_location}->{alignment_score_2} = $alignment_score_2;
|
|
2404 $alignments{$alignment_location}->{sum_of_alignment_scores} = $sum_of_alignment_scores_1;
|
|
2405 $alignments{$alignment_location}->{bowtie_sequence_1} = $bowtie_sequence_1;
|
|
2406 $alignments{$alignment_location}->{bowtie_sequence_2} = $bowtie_sequence_2;
|
|
2407 $alignments{$alignment_location}->{index} = $index;
|
|
2408 $alignments{$alignment_location}->{chromosome} = $chromosome_1; # either is fine
|
|
2409 $alignments{$alignment_location}->{position_1} = $position_1;
|
|
2410 $alignments{$alignment_location}->{position_2} = $position_2;
|
|
2411 $alignments{$alignment_location}->{mismatch_info_1} = $MD_tag_1;
|
|
2412 $alignments{$alignment_location}->{mismatch_info_2} = $MD_tag_2;
|
|
2413 $alignments{$alignment_location}->{CIGAR_1} = $cigar_1;
|
|
2414 $alignments{$alignment_location}->{CIGAR_2} = $cigar_2;
|
|
2415 $alignments{$alignment_location}->{flag_1} = $flag_1;
|
|
2416 $alignments{$alignment_location}->{flag_2} = $flag_2;
|
|
2417 }
|
|
2418
|
|
2419 # warn "added unique alignment to \%alignments hash\n";
|
|
2420
|
|
2421 # Now reading and storing the next read pair
|
|
2422 my $newline_1 = $fhs[$index]->{fh}-> getline();
|
|
2423 my $newline_2 = $fhs[$index]->{fh}-> getline();
|
|
2424 if ($newline_1 and $newline_2){
|
|
2425 chomp $newline_1;
|
|
2426 chomp $newline_2;
|
|
2427 # print "$newline_1\n";
|
|
2428 # print "$newline_2\n";
|
|
2429 my ($seq_id_1) = split (/\t/,$newline_1);
|
|
2430 my ($seq_id_2) = split (/\t/,$newline_2);
|
|
2431 $seq_id_1 =~ s/\/1$//;
|
|
2432 $seq_id_2 =~ s/\/2$//;
|
|
2433 # print "New Seq IDs:\t$seq_id_1\t$seq_id_2\n";
|
|
2434
|
|
2435 $fhs[$index]->{last_seq_id} = $seq_id_1;
|
|
2436 $fhs[$index]->{last_line_1} = $newline_1;
|
|
2437 $fhs[$index]->{last_line_2} = $newline_2;
|
|
2438
|
|
2439 if ($seq_id_1 eq $identifier){
|
|
2440 die "Sequence with ID $identifier did not have a second best alignment, but next seq-ID was also $fhs[$index]->{last_seq_id}!\n";
|
|
2441 }
|
|
2442 }
|
|
2443 else{
|
|
2444 # assigning undef to last_seq_id and last_line_1 and _2 and jumping to the next index (end of Bowtie 2 output)
|
|
2445 $fhs[$index]->{last_seq_id} = undef;
|
|
2446 $fhs[$index]->{last_line_1} = undef;
|
|
2447 $fhs[$index]->{last_line_2} = undef;
|
|
2448 }
|
|
2449 }
|
|
2450 }
|
|
2451 }
|
|
2452
|
|
2453 ### if the read produced several ambiguous alignments for a single instance of Bowtie 2 we can return already now. If --ambiguous was specified the read sequence will be printed out in FastQ format
|
|
2454 if ($alignment_ambiguous == 1){
|
|
2455 $counting{unsuitable_sequence_count}++;
|
|
2456 ### report that the sequence pair has multiple hits with bitwise flag 256. We can print the sequence to the result file straight away and skip everything else
|
|
2457 # my $ambiguous_read_1 = join("\t",$identifier.'/1','256','*','0','0','*','*','0','0',$sequence_1,$quality_value_1);
|
|
2458 # my $ambiguous_read_2 = join("\t",$identifier.'/2','256','*','0','0','*','*','0','0',$sequence_2,$quality_value_2);
|
|
2459 # print "$ambiguous_read_1\n";
|
|
2460 # print "$ambiguous_read_2\n";
|
|
2461
|
|
2462 if ($ambiguous){
|
|
2463 return 2; # => exits to next sequence pair, and prints it out to _ambiguous_reads_1.txt and _ambiguous_reads_2.txt if '--ambiguous' was specified
|
|
2464 }
|
|
2465 elsif ($unmapped){
|
|
2466 return 1; # => exits to next sequence pair, and prints it out to _unmapped_reads_1.txt and _unmapped_reads_2.txt if '--unmapped' but not '--ambiguous' was specified
|
|
2467 }
|
|
2468 else{
|
|
2469 return 0;
|
|
2470 }
|
|
2471 }
|
|
2472
|
|
2473 ### if no alignment was found for a certain sequence at all we continue with the next sequence in the sequence file
|
|
2474 unless (%alignments){
|
|
2475 $counting{no_single_alignment_found}++;
|
|
2476
|
|
2477 # my $unmapped_read_1 = join("\t",$identifier.'/1','77','*','0','0','*','*','0','0',$sequence_1,$quality_value_1);
|
|
2478 # my $unmapped_read_2 = join("\t",$identifier.'/2','141','*','0','0','*','*','0','0',$sequence_2,$quality_value_2);
|
|
2479 # print "$unmapped_read_1\n";
|
|
2480 # print "$unmapped_read_2\n";
|
|
2481 if ($unmapped){
|
|
2482 return 1; # => exits to next sequence pair, and prints it out to _unmapped_reads_1.txt and _unmapped_read_2.txt if '--unmapped' was specified
|
|
2483 }
|
|
2484 else{
|
|
2485 return 0;
|
|
2486 }
|
|
2487 }
|
|
2488
|
|
2489 #######################################################################################################################################################
|
|
2490
|
|
2491 ### If the sequence pair was not rejected so far we are now looking if there is a unique best alignment among all alignment instances. If there is only one
|
|
2492 ### single best position we are going to store the alignment information in the $meth_call variable. If there are multiple hits with the same (highest)
|
|
2493 ### alignment score we are discarding the sequence pair altogether.
|
|
2494 ### For end-to-end alignments the maximum alignment score is 0, each mismatch receives a penalty of 6, and each gap receives penalties for opening (5)
|
|
2495 ### and extending (3 per bp) the gap.
|
|
2496
|
|
2497 #######################################################################################################################################################
|
|
2498
|
|
2499 ### Declaring an empty hash reference which will store all information we need for the methylation call
|
|
2500 my $methylation_call_params; # hash reference
|
|
2501 my $sequence_pair_fails = 0; # using $sequence_pair_fails as a 'memory' if a sequence could not be aligned uniquely (set to 1 then)
|
|
2502
|
|
2503 ### print contents of %alignments for debugging
|
|
2504 ## if (scalar keys %alignments >= 1){
|
|
2505 # print "\n******\n";
|
|
2506 # foreach my $alignment_location (sort {$a cmp $b} keys %alignments){
|
|
2507 # print "Loc: $alignment_location\n";
|
|
2508 # print "ID: $alignments{$alignment_location}->{seq_id}\n";
|
|
2509 # print "AS_1: $alignments{$alignment_location}->{alignment_score_1}\n";
|
|
2510 # print "AS_2: $alignments{$alignment_location}->{alignment_score_2}\n";
|
|
2511 # print "Seq_1: $alignments{$alignment_location}->{bowtie_sequence_1}\n";
|
|
2512 # print "Seq_2: $alignments{$alignment_location}->{bowtie_sequence_2}\n";
|
|
2513 # print "Index $alignments{$alignment_location}->{index}\n";
|
|
2514 # print "Chr: $alignments{$alignment_location}->{chromosome}\n";
|
|
2515 # print "Pos_1: $alignments{$alignment_location}->{position_1}\n";
|
|
2516 # print "Pos_2: $alignments{$alignment_location}->{position_2}\n";
|
|
2517 # print "CIGAR_1: $alignments{$alignment_location}->{CIGAR_1}\n";
|
|
2518 # print "CIGAR_2: $alignments{$alignment_location}->{CIGAR_2}\n";
|
|
2519 # print "MD_1: $alignments{$alignment_location}->{mismatch_info_1}\n";
|
|
2520 # print "MD_2: $alignments{$alignment_location}->{mismatch_info_2}\n";
|
|
2521 # print "Flag 1: $alignments{$alignment_location}->{flag_1}\n";
|
|
2522 # print "Flag 2: $alignments{$alignment_location}->{flag_2}\n";
|
|
2523 # }
|
|
2524 # print "\n******\n";
|
|
2525 # }
|
|
2526
|
|
2527 ### if there is only 1 entry in the %alignments hash we accept it as the best alignment
|
|
2528 if (scalar keys %alignments == 1){
|
|
2529 for my $unique_best_alignment (keys %alignments){
|
|
2530 $methylation_call_params->{$identifier}->{bowtie_sequence_1} = $alignments{$unique_best_alignment}->{bowtie_sequence_1};
|
|
2531 $methylation_call_params->{$identifier}->{bowtie_sequence_2} = $alignments{$unique_best_alignment}->{bowtie_sequence_2};
|
|
2532 $methylation_call_params->{$identifier}->{chromosome} = $alignments{$unique_best_alignment}->{chromosome};
|
|
2533 $methylation_call_params->{$identifier}->{position_1} = $alignments{$unique_best_alignment}->{position_1};
|
|
2534 $methylation_call_params->{$identifier}->{position_2} = $alignments{$unique_best_alignment}->{position_2};
|
|
2535 $methylation_call_params->{$identifier}->{index} = $alignments{$unique_best_alignment}->{index};
|
|
2536 $methylation_call_params->{$identifier}->{alignment_score_1} = $alignments{$unique_best_alignment}->{alignment_score_1};
|
|
2537 $methylation_call_params->{$identifier}->{alignment_score_2} = $alignments{$unique_best_alignment}->{alignment_score_2};
|
|
2538 $methylation_call_params->{$identifier}->{sum_of_alignment_scores} = $alignments{$unique_best_alignment}->{sum_of_alignment_scores};
|
|
2539 $methylation_call_params->{$identifier}->{mismatch_info_1} = $alignments{$unique_best_alignment}->{mismatch_info_1};
|
|
2540 $methylation_call_params->{$identifier}->{mismatch_info_2} = $alignments{$unique_best_alignment}->{mismatch_info_2};
|
|
2541 $methylation_call_params->{$identifier}->{CIGAR_1} = $alignments{$unique_best_alignment}->{CIGAR_1};
|
|
2542 $methylation_call_params->{$identifier}->{CIGAR_2} = $alignments{$unique_best_alignment}->{CIGAR_2};
|
|
2543 $methylation_call_params->{$identifier}->{flag_1} = $alignments{$unique_best_alignment}->{flag_1};
|
|
2544 $methylation_call_params->{$identifier}->{flag_2} = $alignments{$unique_best_alignment}->{flag_2};
|
|
2545 }
|
|
2546 }
|
|
2547
|
|
2548 ### otherwise we are going to find out if there is a best match among the multiple alignments, or whether there are 2 or more equally good alignments (in which case
|
|
2549 ### we boot the sequence pair altogether)
|
|
2550 elsif (scalar keys %alignments >= 2 and scalar keys %alignments <= 4){
|
|
2551 my $best_sum_of_alignment_scores;
|
|
2552 my $best_alignment_location;
|
|
2553 foreach my $alignment_location (sort {$alignments{$b}->{sum_of_alignment_scores} <=> $alignments{$a}->{sum_of_alignment_scores}} keys %alignments){
|
|
2554 # print "$alignments{$alignment_location}->{sum_of_alignment_scores}\n";
|
|
2555 unless (defined $best_sum_of_alignment_scores){
|
|
2556 $best_sum_of_alignment_scores = $alignments{$alignment_location}->{sum_of_alignment_scores};
|
|
2557 $best_alignment_location = $alignment_location;
|
|
2558 # print "setting best alignment score to: $best_sum_of_alignment_scores\n";
|
|
2559 }
|
|
2560 else{
|
|
2561 ### if the second best alignment has the same sum of alignment scores as the first one, the sequence pair will get booted
|
|
2562 if ($alignments{$alignment_location}->{sum_of_alignment_scores} == $best_sum_of_alignment_scores){
|
|
2563 # warn "Same sum of alignment scores for 2 different alignments, the sequence pair will get booted!\n";
|
|
2564 $sequence_pair_fails = 1;
|
|
2565 last; # exiting since we know that the sequence has ambiguous alignments
|
|
2566 }
|
|
2567 ### else we are going to store the best alignment for further processing
|
|
2568 else{
|
|
2569 $methylation_call_params->{$identifier}->{bowtie_sequence_1} = $alignments{$best_alignment_location}->{bowtie_sequence_1};
|
|
2570 $methylation_call_params->{$identifier}->{bowtie_sequence_2} = $alignments{$best_alignment_location}->{bowtie_sequence_2};
|
|
2571 $methylation_call_params->{$identifier}->{chromosome} = $alignments{$best_alignment_location}->{chromosome};
|
|
2572 $methylation_call_params->{$identifier}->{position_1} = $alignments{$best_alignment_location}->{position_1};
|
|
2573 $methylation_call_params->{$identifier}->{position_2} = $alignments{$best_alignment_location}->{position_2};
|
|
2574 $methylation_call_params->{$identifier}->{index} = $alignments{$best_alignment_location}->{index};
|
|
2575 $methylation_call_params->{$identifier}->{alignment_score_1} = $alignments{$best_alignment_location}->{alignment_score_1};
|
|
2576 $methylation_call_params->{$identifier}->{alignment_score_2} = $alignments{$best_alignment_location}->{alignment_score_2};
|
|
2577 $methylation_call_params->{$identifier}->{sum_of_alignment_scores} = $alignments{$best_alignment_location}->{sum_of_alignment_scores};
|
|
2578 $methylation_call_params->{$identifier}->{mismatch_info_1} = $alignments{$best_alignment_location}->{mismatch_info_1};
|
|
2579 $methylation_call_params->{$identifier}->{mismatch_info_2} = $alignments{$best_alignment_location}->{mismatch_info_2};
|
|
2580 $methylation_call_params->{$identifier}->{CIGAR_1} = $alignments{$best_alignment_location}->{CIGAR_1};
|
|
2581 $methylation_call_params->{$identifier}->{CIGAR_2} = $alignments{$best_alignment_location}->{CIGAR_2};
|
|
2582 $methylation_call_params->{$identifier}->{flag_1} = $alignments{$best_alignment_location}->{flag_1};
|
|
2583 $methylation_call_params->{$identifier}->{flag_2} = $alignments{$best_alignment_location}->{flag_2};
|
|
2584 last; # exiting since the sequence produced a unique best alignment
|
|
2585 }
|
|
2586 }
|
|
2587 }
|
|
2588 }
|
|
2589 else{
|
|
2590 die "There are too many potential hits for this sequence pair (1-4 expected, but found: '",scalar keys %alignments,"')\n";;
|
|
2591 }
|
|
2592
|
|
2593 ### skipping the sequence completely if there were multiple alignments with the same best sum of alignment scores at different positions
|
|
2594 if ($sequence_pair_fails == 1){
|
|
2595 $counting{unsuitable_sequence_count}++;
|
|
2596
|
|
2597 ### report that the sequence has multiple hits with bitwise flag 256. We can print the sequence to the result file straight away and skip everything else
|
|
2598 # my $ambiguous_read_1 = join("\t",$identifier.'/1','256','*','0','0','*','*','0','0',$sequence_1,$quality_value_1);
|
|
2599 # my $ambiguous_read_2 = join("\t",$identifier.'/2','256','*','0','0','*','*','0','0',$sequence_2,$quality_value_2);
|
|
2600 # print "$ambiguous_read_1\n";
|
|
2601 # print "$ambiguous_read_2\n";
|
|
2602
|
|
2603 if ($ambiguous){
|
|
2604 return 2; # => exits to next sequence pair, and prints it out (in FastQ format) to _ambiguous_reads_1.txt and _ambiguous_reads_2.txt if '--ambiguous' was specified
|
|
2605 }
|
|
2606 elsif ($unmapped){
|
|
2607 return 1; # => exits to next sequence pair, and prints it out (in FastQ format) to _unmapped_reads_1.txt and _unmapped_reads_2.txt if '--unmapped' but not '--ambiguous' was specified
|
|
2608 }
|
|
2609 else{
|
|
2610 return 0; # => exits to next sequence pair (default)
|
|
2611 }
|
|
2612 }
|
|
2613
|
|
2614 ### --DIRECTIONAL
|
|
2615 ### If the option --directional has been specified the user wants to consider only alignments to the original top strand or the original bottom strand. We will therefore
|
|
2616 ### discard all alignments to strands complementary to the original strands, as they should not exist in reality due to the library preparation protocol
|
|
2617 if ($directional){
|
|
2618 if ( ($methylation_call_params->{$identifier}->{index} == 1) or ($methylation_call_params->{$identifier}->{index} == 2) ){
|
|
2619 # warn "Alignment rejected! (index was: $methylation_call_params->{$identifier}->{index})\n";
|
|
2620 $counting{alignments_rejected_count}++;
|
|
2621 return 0;
|
|
2622 }
|
|
2623 }
|
|
2624
|
|
2625 ### If the sequence pair has not been rejected so far it does have a unique best alignment
|
|
2626 $counting{unique_best_alignment_count}++;
|
|
2627 extract_corresponding_genomic_sequence_paired_ends_bowtie2($identifier,$methylation_call_params);
|
|
2628
|
|
2629 ### check to see if the genomic sequences we extracted has the same length as the observed sequences +2, and only then we perform the methylation call
|
|
2630 if (length($methylation_call_params->{$identifier}->{unmodified_genomic_sequence_1}) != length($sequence_1)+2){
|
|
2631 warn "Chromosomal sequence could not be extracted for\t$identifier\t$methylation_call_params->{$identifier}->{chromosome}\t$methylation_call_params->{$identifier}->{start_seq_1}\n";
|
|
2632 $counting{genomic_sequence_could_not_be_extracted_count}++;
|
|
2633 return 0;
|
|
2634 }
|
|
2635 if (length($methylation_call_params->{$identifier}->{unmodified_genomic_sequence_2}) != length($sequence_2)+2){
|
|
2636 warn "Chromosomal sequence could not be extracted for\t$identifier\t$methylation_call_params->{$identifier}->{chromosome}\t$methylation_call_params->{$identifier}->{start_seq_2}\n";
|
|
2637 $counting{genomic_sequence_could_not_be_extracted_count}++;
|
|
2638 return 0;
|
|
2639 }
|
|
2640
|
|
2641 ### now we are set to perform the actual methylation call
|
|
2642 $methylation_call_params->{$identifier}->{methylation_call_1} = methylation_call($identifier,$sequence_1,$methylation_call_params->{$identifier}->{unmodified_genomic_sequence_1},$methylation_call_params->{$identifier}->{read_conversion_1});
|
|
2643 $methylation_call_params->{$identifier}->{methylation_call_2} = methylation_call($identifier,$sequence_2,$methylation_call_params->{$identifier}->{unmodified_genomic_sequence_2},$methylation_call_params->{$identifier}->{read_conversion_2});
|
|
2644 # print "$methylation_call_params->{$identifier}->{read_conversion_2}\n";
|
|
2645 # print " $sequence_2\n";
|
|
2646 # print "$methylation_call_params->{$identifier}->{unmodified_genomic_sequence_2}\n";
|
|
2647 # print " $methylation_call_params->{$identifier}->{methylation_call_2}\n";
|
|
2648
|
|
2649 print_bisulfite_mapping_results_paired_ends_bowtie2($identifier,$sequence_1,$sequence_2,$methylation_call_params,$quality_value_1,$quality_value_2);
|
|
2650 return 0; ## otherwise 1 will be returned by default, which would print the sequence pair to unmapped_1 and _2
|
|
2651 }
|
|
2652
|
|
2653 ###
|
|
2654
|
|
2655 sub decide_whether_paired_end_alignment_is_valid{
|
|
2656 my ($index,$identifier) = @_;
|
|
2657 my ($id_1,$strand_1,$mapped_chromosome_1,$position_1,$bowtie_sequence_1,$mismatch_info_1) = (split (/\t/,$fhs[$index]->{last_line_1},-1))[0,1,2,3,4,7];
|
|
2658 my ($id_2,$strand_2,$mapped_chromosome_2,$position_2,$bowtie_sequence_2,$mismatch_info_2) = (split (/\t/,$fhs[$index]->{last_line_2},-1))[0,1,2,3,4,7];
|
|
2659 chomp $mismatch_info_1;
|
|
2660 chomp $mismatch_info_2;
|
|
2661 my $seq_id_1 = $id_1;
|
|
2662 my $seq_id_2 = $id_2;
|
|
2663 $seq_id_1 =~ s/\/1$//; # removing the read /1
|
|
2664 $seq_id_2 =~ s/\/1$//; # removing the read /1
|
|
2665
|
|
2666 ### ensuring that the current entry is the correct sequence
|
|
2667 if ($seq_id_1 eq $identifier or $seq_id_2 eq $identifier){
|
|
2668 ### checking the orientation of the alignment. We need to discriminate between 8 different conditions, however only 4 of them are theoretically
|
|
2669 ### sensible alignments
|
|
2670 my $orientation = ensure_sensical_alignment_orientation_paired_ends ($index,$id_1,$strand_1,$id_2,$strand_2);
|
|
2671 ### If the orientation was correct can we move on
|
|
2672 if ($orientation == 1){
|
|
2673 return 1; ### 1st possibility for A SEQUENCE-PAIR TO PASS
|
|
2674 }
|
|
2675 ### If the alignment was in the wrong orientation we need to read in two new lines
|
|
2676 elsif($orientation == 0){
|
|
2677 my $newline_1 = $fhs[$index]->{fh}->getline();
|
|
2678 my $newline_2 = $fhs[$index]->{fh}->getline();
|
|
2679 if ($newline_1 and $newline_2){
|
|
2680 ### extract detailed information about the alignment again (from $newline_1 and $newline_2 this time)
|
|
2681 ($id_1,$strand_1) = (split (/\t/,$newline_1))[0,1];
|
|
2682 ($id_2,$strand_2) = (split (/\t/,$newline_2))[0,1];
|
|
2683
|
|
2684 my $seqid;
|
|
2685 $seq_id_1 = $id_1;
|
|
2686 $seq_id_2 = $id_2;
|
|
2687 # we need to capture the first read (ending on /1)
|
|
2688 if ($seq_id_1 =~ s/\/1$//){ # removing the read /1 tag
|
|
2689 $seqid = $seq_id_1;
|
|
2690 }
|
|
2691 elsif ($seq_id_2 =~ s/\/1$//){ # removing the read /1 tag
|
|
2692 $seqid = $seq_id_2;
|
|
2693 }
|
|
2694 else{
|
|
2695 die "One of the two reads needs to end on /1!!";
|
|
2696 }
|
|
2697
|
|
2698 ### ensuring that the next entry is still the correct sequence
|
|
2699 if ($seq_id_1 eq $identifier or $seq_id_2 eq $identifier){
|
|
2700 ### checking orientation again
|
|
2701 $orientation = ensure_sensical_alignment_orientation_paired_ends ($index,$id_1,$strand_1,$id_2,$strand_2);
|
|
2702 ### If the orientation was correct can we move on
|
|
2703 if ($orientation == 1){
|
|
2704 ### Writing the current sequence to last_line_1 and last_line_2
|
|
2705 $fhs[$index]->{last_seq_id} = $seqid;
|
|
2706 $fhs[$index]->{last_line_1} = $newline_1;
|
|
2707 $fhs[$index]->{last_line_2} = $newline_2;
|
|
2708 return 1; ### 2nd possibility for a SEQUENCE-PAIR TO PASS
|
|
2709 }
|
|
2710 ### If the alignment was in the wrong orientation again we need to read in yet another 2 new lines and store them in @fhs (this must be
|
|
2711 ### the next entry)
|
|
2712 elsif ($orientation == 0){
|
|
2713 $newline_1 = $fhs[$index]->{fh}->getline();
|
|
2714 $newline_2 = $fhs[$index]->{fh}->getline();
|
|
2715 if ($newline_1 and $newline_2){
|
|
2716 ($seq_id_1) = split (/\t/,$newline_1);
|
|
2717 ($seq_id_2) = split (/\t/,$newline_2);
|
|
2718
|
|
2719 $seqid = '';
|
|
2720 if ($seq_id_1 =~ s/\/1$//){ # removing the read /1 tag
|
|
2721 $seqid = $seq_id_1;
|
|
2722 }
|
|
2723 elsif ($seq_id_2 =~ s/\/1$//){ # removing the read /1 tag
|
|
2724 $seqid = $seq_id_2;
|
|
2725 }
|
|
2726 else{
|
|
2727 die "One of the two reads needs to end on /1!!";
|
|
2728 }
|
|
2729
|
|
2730 ### check if the next 2 lines still have the same seq ID (must not happen), and if not overwrite the current seq-ID and bowtie output with
|
|
2731 ### the same fields of the just read next entry
|
|
2732 die "Same seq ID 3 or more times in a row!(should be 2 max)" if ($seqid eq $identifier);
|
|
2733 $fhs[$index]->{last_seq_id} = $seqid;
|
|
2734 $fhs[$index]->{last_line_1} = $newline_1;
|
|
2735 $fhs[$index]->{last_line_2} = $newline_2;
|
|
2736 return 0; # not processing anything this round as the alignment currently stored in last_line_1 and _2 was in the wrong orientation
|
|
2737 }
|
|
2738 else {
|
|
2739 ### assigning undef to last_seq_id and last_line (end of bowtie output)
|
|
2740 $fhs[$index]->{last_seq_id} = undef;
|
|
2741 $fhs[$index]->{last_line_1} = undef;
|
|
2742 $fhs[$index]->{last_line_2} = undef;
|
|
2743 return 0; # not processing anything as the alignment currently stored in last_line_1 and _2 was in the wrong orientation
|
|
2744 }
|
|
2745 }
|
|
2746 else{
|
|
2747 die "The orientation of the alignment must be either correct or incorrect\n";
|
|
2748 }
|
|
2749 }
|
|
2750 ### the sequence pair we just read in is already the next sequence pair to be analysed -> store it in @fhs
|
|
2751 else{
|
|
2752 $fhs[$index]->{last_seq_id} = $seqid;
|
|
2753 $fhs[$index]->{last_line_1} = $newline_1;
|
|
2754 $fhs[$index]->{last_line_2} = $newline_2;
|
|
2755 return 0; # processing the new alignment result only in the next round
|
|
2756 }
|
|
2757 }
|
|
2758 else {
|
|
2759 # assigning undef to last_seq_id and both last_lines (end of bowtie output)
|
|
2760 $fhs[$index]->{last_seq_id} = undef;
|
|
2761 $fhs[$index]->{last_line_1} = undef;
|
|
2762 $fhs[$index]->{last_line_2} = undef;
|
|
2763 return 0; # not processing anything as the alignment currently stored in last_line_1 and _2 was in the wrong orientation
|
|
2764 }
|
|
2765 }
|
|
2766 else{
|
|
2767 die "The orientation of the alignment must be either correct or incorrect\n";
|
|
2768 }
|
|
2769 }
|
|
2770 ### the sequence pair stored in @fhs as last_line_1 and last_line_2 is already the next sequence pair to be analysed -> analyse next round
|
|
2771 else{
|
|
2772 return 0;
|
|
2773 }
|
|
2774 }
|
|
2775
|
|
2776 ### EXTRACT GENOMIC SEQUENCE | BOWTIE 1 | PAIRED-END
|
|
2777
|
|
2778 sub extract_corresponding_genomic_sequence_paired_ends {
|
|
2779 my ($sequence_identifier,$methylation_call_params) = @_;
|
|
2780 ### A bisulfite sequence pair for 1 location in the genome can theoretically be on any of the 4 possible converted strands. We are also giving the
|
|
2781 ### sequence a 'memory' of the conversion we are expecting which we will need later for the methylation call
|
|
2782 my $alignment_read_1;
|
|
2783 my $alignment_read_2;
|
|
2784 my $read_conversion_info_1;
|
|
2785 my $read_conversion_info_2;
|
|
2786 my $genome_conversion;
|
|
2787
|
|
2788 ### Now extracting the same sequence from the mouse genomic sequence, +2 extra bases at oone of the ends so that we can also make a CpG, CHG or CHH methylation call
|
|
2789 ### if the C happens to be at the first or last position of the actually observed sequence
|
|
2790 my $non_bisulfite_sequence_1;
|
|
2791 my $non_bisulfite_sequence_2;
|
|
2792
|
|
2793 ### all alignments reported by bowtie have the + alignment first and the - alignment as the second one irrespective of whether read 1 or read 2 was
|
|
2794 ### the + alignment. We however always read in sequences read 1 then read 2, so if read 2 is the + alignment we need to swap the extracted genomic
|
|
2795 ### sequences around!
|
|
2796 ### results from CT converted read 1 plus GA converted read 2 vs. CT converted genome (+/- orientation alignments are reported only)
|
|
2797 if ($methylation_call_params->{$sequence_identifier}->{index} == 0){
|
|
2798 ### [Index 0, sequence originated from (converted) forward strand]
|
|
2799 $counting{CT_GA_CT_count}++;
|
|
2800 $alignment_read_1 = '+';
|
|
2801 $alignment_read_2 = '-';
|
|
2802 $read_conversion_info_1 = 'CT';
|
|
2803 $read_conversion_info_2 = 'GA';
|
|
2804 $genome_conversion = 'CT';
|
|
2805 ### SEQUENCE 1 (this is always the forward hit, in this case it is read 1)
|
|
2806 ### for hits on the forward strand we need to capture 2 extra bases at the 3' end
|
|
2807
|
|
2808 $non_bisulfite_sequence_1 = substr ($chromosomes{$methylation_call_params->{$sequence_identifier}->{chromosome}},$methylation_call_params->{$sequence_identifier}->{start_seq_1},length($methylation_call_params->{$sequence_identifier}->{bowtie_sequence_1})+2); ##CHH change
|
|
2809
|
|
2810 ### SEQUENCE 2 (this will always be on the reverse strand, in this case it is read 2)
|
|
2811 ### As the second conversion is GA we need to capture 1 base 3', so that it is a 5' base after reverse complementation
|
|
2812 if (length($chromosomes{$methylation_call_params->{$sequence_identifier}->{chromosome}}) > $methylation_call_params->{$sequence_identifier}->{start_seq_2}+length($methylation_call_params->{$sequence_identifier}->{bowtie_sequence_2})+1){ ## CHH change to +1
|
|
2813
|
|
2814 $non_bisulfite_sequence_2 = substr ($chromosomes{$methylation_call_params->{$sequence_identifier}->{chromosome}},($methylation_call_params->{$sequence_identifier}->{start_seq_2}),length($methylation_call_params->{$sequence_identifier}->{bowtie_sequence_2})+2);
|
|
2815 ### the reverse strand sequence needs to be reverse complemented
|
|
2816 $non_bisulfite_sequence_2 = reverse_complement($non_bisulfite_sequence_2);
|
|
2817 }
|
|
2818 else{
|
|
2819 $non_bisulfite_sequence_2 = '';
|
|
2820 }
|
|
2821 }
|
|
2822
|
|
2823 ### results from GA converted read 1 plus CT converted read 2 vs. GA converted genome (+/- orientation alignments are reported only)
|
|
2824 elsif ($methylation_call_params->{$sequence_identifier}->{index} == 1){
|
|
2825 ### [Index 1, sequence originated from complementary to (converted) reverse strand]
|
|
2826 $counting{GA_CT_GA_count}++;
|
|
2827 $alignment_read_1 = '+';
|
|
2828 $alignment_read_2 = '-';
|
|
2829 $read_conversion_info_1 = 'GA';
|
|
2830 $read_conversion_info_2 = 'CT';
|
|
2831 $genome_conversion = 'GA';
|
|
2832
|
|
2833 ### SEQUENCE 1 (this is always the forward hit, in this case it is read 1)
|
|
2834 ### as we need to make the methylation call for the base 5' of the first base (GA conversion!) we need to capture 2 extra bases at the 5' end
|
|
2835 if ($methylation_call_params->{$sequence_identifier}->{start_seq_1}-1 > 0){ ## CHH change to -1
|
|
2836 $non_bisulfite_sequence_1 = substr ($chromosomes{$methylation_call_params->{$sequence_identifier}->{chromosome}},$methylation_call_params->{$sequence_identifier}->{start_seq_1}-2,length($methylation_call_params->{$sequence_identifier}->{bowtie_sequence_1})+2); ### CHH change to -2/+2
|
|
2837 }
|
|
2838 else{
|
|
2839 $non_bisulfite_sequence_1 = '';
|
|
2840 }
|
|
2841
|
|
2842 ### SEQUENCE 2 (this will always be on the reverse strand, in this case it is read 2)
|
|
2843 ### As we are doing a CT comparison for the reverse strand we are taking 2 bases extra at the 5' end, so it is a 3' base after reverse complementation
|
|
2844 $non_bisulfite_sequence_2 = substr ($chromosomes{$methylation_call_params->{$sequence_identifier}->{chromosome}},($methylation_call_params->{$sequence_identifier}->{start_seq_2})-2,length($methylation_call_params->{$sequence_identifier}->{bowtie_sequence_2})+2); ### CHH change to -2/+2
|
|
2845 ### the reverse strand sequence needs to be reverse complemented
|
|
2846 $non_bisulfite_sequence_2 = reverse_complement($non_bisulfite_sequence_2);
|
|
2847 }
|
|
2848
|
|
2849 ### results from GA converted read 1 plus CT converted read 2 vs. CT converted genome (-/+ orientation alignments are reported only)
|
|
2850 elsif ($methylation_call_params->{$sequence_identifier}->{index} == 2){
|
|
2851 ### [Index 2, sequence originated from the complementary to (converted) forward strand]
|
|
2852 $counting{GA_CT_CT_count}++;
|
|
2853 $alignment_read_1 = '-';
|
|
2854 $alignment_read_2 = '+';
|
|
2855 $read_conversion_info_1 = 'GA';
|
|
2856 $read_conversion_info_2 = 'CT';
|
|
2857 $genome_conversion = 'CT';
|
|
2858
|
|
2859 ### Here we switch the sequence information round!! non_bisulfite_sequence_1 will later correspond to the read 1!!!!
|
|
2860 ### SEQUENCE 1 (this is always the forward hit, in this case it is READ 2), read 1 is in - orientation on the reverse strand
|
|
2861 ### As read 1 is GA converted we need to capture 2 extra 3' bases which will be 2 extra 5' base after reverse complementation
|
|
2862 $non_bisulfite_sequence_1 = substr ($chromosomes{$methylation_call_params->{$sequence_identifier}->{chromosome}},($methylation_call_params->{$sequence_identifier}->{start_seq_2}),length($methylation_call_params->{$sequence_identifier}->{bowtie_sequence_2})+2); ### CHH change to +2
|
|
2863 ### the reverse strand sequence needs to be reverse complemented
|
|
2864 $non_bisulfite_sequence_1 = reverse_complement($non_bisulfite_sequence_1);
|
|
2865
|
|
2866 ### SEQUENCE 2 (this will always be on the reverse strand, in this case it is READ 1)
|
|
2867 ### non_bisulfite_sequence_2 will later correspond to the read 2!!!!
|
|
2868 ### Read 2 is CT converted so we need to capture 2 extra 3' bases
|
|
2869 if (length($chromosomes{$methylation_call_params->{$sequence_identifier}->{chromosome}}) > ($methylation_call_params->{$sequence_identifier}->{start_seq_1})+length($methylation_call_params->{$sequence_identifier}->{bowtie_sequence_1})+1){ ## CHH change to +1
|
|
2870 $non_bisulfite_sequence_2 = substr ($chromosomes{$methylation_call_params->{$sequence_identifier}->{chromosome}},($methylation_call_params->{$sequence_identifier}->{start_seq_1}),length($methylation_call_params->{$sequence_identifier}->{bowtie_sequence_1})+2); ## CHH changed from +1 to +2
|
|
2871 }
|
|
2872 else{
|
|
2873 $non_bisulfite_sequence_2 = '';
|
|
2874 }
|
|
2875 }
|
|
2876
|
|
2877 ### results from CT converted read 1 plus GA converted read 2 vs. GA converted genome (-/+ orientation alignments are reported only)
|
|
2878 elsif ($methylation_call_params->{$sequence_identifier}->{index} == 3){
|
|
2879 ### [Index 3, sequence originated from the (converted) reverse strand]
|
|
2880 $counting{CT_GA_GA_count}++;
|
|
2881 $alignment_read_1 = '-';
|
|
2882 $alignment_read_2 = '+';
|
|
2883 $read_conversion_info_1 = 'CT';
|
|
2884 $read_conversion_info_2 = 'GA';
|
|
2885 $genome_conversion = 'GA';
|
|
2886
|
|
2887 ### Here we switch the sequence information round!! non_bisulfite_sequence_1 will later correspond to the read 1!!!!
|
|
2888 ### SEQUENCE 1 (this is always the forward hit, in this case it is READ 2), read 1 is in - orientation on the reverse strand
|
|
2889 ### As read 1 is CT converted we need to capture 2 extra 5' bases which will be 2 extra 3' base after reverse complementation
|
|
2890 if ( ($methylation_call_params->{$sequence_identifier}->{start_seq_2}-1) > 0){ ## CHH changed to -1
|
|
2891 $non_bisulfite_sequence_1 = substr ($chromosomes{$methylation_call_params->{$sequence_identifier}->{chromosome}},($methylation_call_params->{$sequence_identifier}->{start_seq_2})-2,length($methylation_call_params->{$sequence_identifier}->{bowtie_sequence_2})+2); ### CHH changed to -2/+2
|
|
2892 ### the reverse strand sequence needs to be reverse complemented
|
|
2893 $non_bisulfite_sequence_1 = reverse_complement($non_bisulfite_sequence_1);
|
|
2894 }
|
|
2895 else{
|
|
2896 $non_bisulfite_sequence_1 = '';
|
|
2897 }
|
|
2898
|
|
2899 ### SEQUENCE 2 (this will always be on the reverse strand, in this case it is READ 1)
|
|
2900 ### non_bisulfite_sequence_2 will later correspond to the read 2!!!!
|
|
2901 ### Read 2 is GA converted so we need to capture 2 extra 5' bases
|
|
2902 $non_bisulfite_sequence_2 = substr ($chromosomes{$methylation_call_params->{$sequence_identifier}->{chromosome}},($methylation_call_params->{$sequence_identifier}->{start_seq_1})-2,length($methylation_call_params->{$sequence_identifier}->{bowtie_sequence_1})+2); ### CHH changed to -2/+2
|
|
2903 }
|
|
2904 else{
|
|
2905 die "Too many bowtie result filehandles\n";
|
|
2906 }
|
|
2907 ### the alignment_strand information is needed to determine which strand of the genomic sequence we are comparing the read against,
|
|
2908 ### the read_conversion information is needed to know whether we are looking for C->T or G->A substitutions
|
|
2909
|
|
2910 $methylation_call_params->{$sequence_identifier}->{alignment_read_1} = $alignment_read_1;
|
|
2911 $methylation_call_params->{$sequence_identifier}->{alignment_read_2} = $alignment_read_2;
|
|
2912 $methylation_call_params->{$sequence_identifier}->{genome_conversion} = $genome_conversion;
|
|
2913 $methylation_call_params->{$sequence_identifier}->{read_conversion_1} = $read_conversion_info_1;
|
|
2914 $methylation_call_params->{$sequence_identifier}->{read_conversion_2} = $read_conversion_info_2;
|
|
2915 $methylation_call_params->{$sequence_identifier}->{unmodified_genomic_sequence_1} = $non_bisulfite_sequence_1;
|
|
2916 $methylation_call_params->{$sequence_identifier}->{unmodified_genomic_sequence_2} = $non_bisulfite_sequence_2;
|
|
2917 }
|
|
2918
|
|
2919 ### EXTRACT GENOMIC SEQUENCE BOWTIE 2 | PAIRED-END
|
|
2920
|
|
2921 sub extract_corresponding_genomic_sequence_paired_ends_bowtie2{
|
|
2922 my ($sequence_identifier,$methylation_call_params) = @_;
|
|
2923 ### A bisulfite sequence pair for 1 location in the genome can theoretically be on any of the 4 possible converted strands. We are also giving the
|
|
2924 ### sequence a 'memory' of the conversion we are expecting which we will need later for the methylation call
|
|
2925
|
|
2926 my $cigar_1 = $methylation_call_params->{$sequence_identifier}->{CIGAR_1};
|
|
2927 my $cigar_2 = $methylation_call_params->{$sequence_identifier}->{CIGAR_2};
|
|
2928 my $flag_1 = $methylation_call_params->{$sequence_identifier}->{flag_1};
|
|
2929 my $flag_2 = $methylation_call_params->{$sequence_identifier}->{flag_2};
|
|
2930 # print "$cigar_1\t$cigar_2\t$flag_1\t$flag_2\n";
|
|
2931 ### We are now extracting the corresponding genomic sequence, +2 extra bases at the end (or start) so that we can also make a CpG methylation call and
|
|
2932 ### in addition make differential calls for Cs in CHG or CHH context if the C happens to be at the last (or first) position of the actually observed sequence
|
|
2933
|
|
2934 ### the alignment_strand information is needed to determine which strand of the genomic sequence we are comparing the read against,
|
|
2935 ### the read_conversion information is needed to know whether we are looking for C->T or G->A substitutions
|
|
2936 my $alignment_read_1;
|
|
2937 my $alignment_read_2;
|
|
2938 my $read_conversion_info_1;
|
|
2939 my $read_conversion_info_2;
|
|
2940 my $genome_conversion;
|
|
2941
|
|
2942 ### Now extracting the same sequence from the mouse genomic sequence, +2 extra bases at one of the ends so that we can also make a CpG, CHG or CHH methylation call
|
|
2943 ### if the C happens to be at the last position of the actually observed sequence
|
|
2944 my $non_bisulfite_sequence_1 = '';
|
|
2945 my $non_bisulfite_sequence_2 = '';
|
|
2946
|
|
2947 ### Positions in SAM format are 1 based, so we need to subract 1 when getting substrings
|
|
2948 my $pos_1 = $methylation_call_params->{$sequence_identifier}->{position_1}-1;
|
|
2949 my $pos_2 = $methylation_call_params->{$sequence_identifier}->{position_2}-1;
|
|
2950
|
|
2951 # parsing CIGAR 1 string
|
|
2952 my @len_1 = split (/\D+/,$cigar_1); # storing the length per operation
|
|
2953 my @ops_1 = split (/\d+/,$cigar_1); # storing the operation
|
|
2954 shift @ops_1; # remove the empty first element
|
|
2955 die "CIGAR 1 string contained a non-matching number of lengths and operations\n" unless (scalar @len_1 == scalar @ops_1);
|
|
2956 # parsing CIGAR 2 string
|
|
2957 my @len_2 = split (/\D+/,$cigar_2); # storing the length per operation
|
|
2958 my @ops_2 = split (/\d+/,$cigar_2); # storing the operation
|
|
2959 shift @ops_2; # remove the empty first element
|
|
2960 die "CIGAR 2 string contained a non-matching number of lengths and operations\n" unless (scalar @len_2 == scalar @ops_2);
|
|
2961
|
|
2962 my $indels_1 = 0; # addiong these to the hemming distance value (needed for the NM field in the final SAM output
|
|
2963 my $indels_2 = 0;
|
|
2964
|
|
2965 ### Extracting read 1 genomic sequence ###
|
|
2966
|
|
2967 # extracting 2 additional bp at the 5' end (read 1)
|
|
2968 if ( ($methylation_call_params->{$sequence_identifier}->{index} == 1) or ($methylation_call_params->{$sequence_identifier}->{index} == 3) ){
|
|
2969 # checking if the substring will be valid or if we can't extract the sequence because we are right at the edge of a chromosome
|
|
2970 unless ( ($pos_1-2) > 0){# exiting with en empty genomic sequence otherwise
|
|
2971 $methylation_call_params->{$sequence_identifier}->{unmodified_genomic_sequence_1} = $non_bisulfite_sequence_1;
|
|
2972 return;
|
|
2973 }
|
|
2974 $non_bisulfite_sequence_1 .= substr ($chromosomes{$methylation_call_params->{$sequence_identifier}->{chromosome}},$pos_1-2,2);
|
|
2975 }
|
|
2976
|
|
2977 foreach (0..$#len_1){
|
|
2978 if ($ops_1[$_] eq 'M'){
|
|
2979 # extracting genomic sequence
|
|
2980 $non_bisulfite_sequence_1 .= substr ($chromosomes{$methylation_call_params->{$sequence_identifier}->{chromosome}},$pos_1,$len_1[$_]);
|
|
2981 # warn "$non_bisulfite_sequence_1\n";
|
|
2982 # adjusting position
|
|
2983 $pos_1 += $len_1[$_];
|
|
2984 }
|
|
2985 elsif ($ops_1[$_] eq 'I'){ # insertion in the read sequence
|
|
2986 # we simply add padding Ns instead of finding genomic sequence. This will not be used to infer methylation calls
|
|
2987 $non_bisulfite_sequence_1 .= 'N' x $len_1[$_];
|
|
2988 # warn "$non_bisulfite_sequence_1\n";
|
|
2989 # position doesn't need adjusting
|
|
2990 $indels_1 += $len_1[$_]; # adding to $indels_1 to determine the hemming distance (= single base mismatches, insertions or deletions) for the SAM output
|
|
2991 }
|
|
2992 elsif ($ops_1[$_] eq 'D'){ # deletion in the read sequence
|
|
2993 # we do not add any genomic sequence but only adjust the position
|
|
2994 # warn "Just adjusting the position by: ",$len_1[$_],"bp\n";
|
|
2995 $pos_1 += $len_1[$_];
|
|
2996 $indels_1 += $len_1[$_]; # adding to $indels_1 to determine the hemming distance (= single base mismatches, insertions or deletions) for the SAM output
|
|
2997 }
|
|
2998 elsif($cigar_1 =~ tr/[NSHPX=]//){ # if these (for standard mapping) illegal characters exist we die
|
|
2999 die "The CIGAR 1 string contained illegal CIGAR operations in addition to 'M', 'I' and 'D': $cigar_1\n";
|
|
3000 }
|
|
3001 else{
|
|
3002 die "The CIGAR 1 string contained undefined CIGAR operations in addition to 'M', 'I' and 'D': $cigar_1\n";
|
|
3003 }
|
|
3004 }
|
|
3005
|
|
3006 ### 3' end of read 1
|
|
3007 if ( ($methylation_call_params->{$sequence_identifier}->{index} == 0) or ($methylation_call_params->{$sequence_identifier}->{index} == 2) ){
|
|
3008 ## checking if the substring will be valid or if we can't extract the sequence because we are right at the edge of a chromosome
|
|
3009 unless (length($chromosomes{$methylation_call_params->{$sequence_identifier}->{chromosome}}) >= $pos_1+2){# exiting with en empty genomic sequence otherwise
|
|
3010 $methylation_call_params->{$sequence_identifier}->{unmodified_genomic_sequence_1} = $non_bisulfite_sequence_1;
|
|
3011 return;
|
|
3012 }
|
|
3013 $non_bisulfite_sequence_1 .= substr ($chromosomes{$methylation_call_params->{$sequence_identifier}->{chromosome}},$pos_1,2);
|
|
3014 }
|
|
3015
|
|
3016
|
|
3017 ### Extracting read 2 genomic sequence ###
|
|
3018
|
|
3019 ### 5' end of read 2
|
|
3020 if ( ($methylation_call_params->{$sequence_identifier}->{index} == 1) or ($methylation_call_params->{$sequence_identifier}->{index} == 3) ){
|
|
3021 ## checking if the substring will be valid or if we can't extract the sequence because we are right at the edge of a chromosome
|
|
3022 unless ( ($pos_2-2) >= 0){# exiting with en empty genomic sequence otherwise
|
|
3023 $methylation_call_params->{$sequence_identifier}->{unmodified_genomic_sequence_2} = $non_bisulfite_sequence_2;
|
|
3024 return;
|
|
3025 }
|
|
3026 $non_bisulfite_sequence_2 .= substr ($chromosomes{$methylation_call_params->{$sequence_identifier}->{chromosome}},$pos_2-2,2);
|
|
3027 }
|
|
3028
|
|
3029 foreach (0..$#len_2){
|
|
3030 if ($ops_2[$_] eq 'M'){
|
|
3031 # extracting genomic sequence
|
|
3032 $non_bisulfite_sequence_2 .= substr ($chromosomes{$methylation_call_params->{$sequence_identifier}->{chromosome}},$pos_2,$len_2[$_]);
|
|
3033 # warn "$non_bisulfite_sequence_2\n";
|
|
3034 # adjusting position
|
|
3035 $pos_2 += $len_2[$_];
|
|
3036 }
|
|
3037 elsif ($ops_2[$_] eq 'I'){ # insertion in the read sequence
|
|
3038 # we simply add padding Ns instead of finding genomic sequence. This will not be used to infer methylation calls
|
|
3039 $non_bisulfite_sequence_2 .= 'N' x $len_2[$_];
|
|
3040 # warn "$non_bisulfite_sequence_2\n";
|
|
3041 # position doesn't need adjusting
|
|
3042 $indels_2 += $len_2[$_]; # adding to $indels_1 to determine the hemming distance (= single base mismatches, insertions or deletions) for the SAM output
|
|
3043 }
|
|
3044 elsif ($ops_2[$_] eq 'D'){ # deletion in the read sequence
|
|
3045 # we do not add any genomic sequence but only adjust the position
|
|
3046 # warn "Just adjusting the position by: ",$len_2[$_],"bp\n";
|
|
3047 $pos_2 += $len_2[$_];
|
|
3048 $indels_2 += $len_2[$_]; # adding to $indels_1 to determine the hemming distance (= single base mismatches, insertions or deletions) for the SAM output
|
|
3049 }
|
|
3050 elsif($cigar_2 =~ tr/[NSHPX=]//){ # if these (for standard mapping) illegal characters exist we die
|
|
3051 die "The CIGAR 2 string contained illegal CIGAR operations in addition to 'M', 'I' and 'D': $cigar_2\n";
|
|
3052 }
|
|
3053 else{
|
|
3054 die "The CIGAR 2 string contained undefined CIGAR operations in addition to 'M', 'I' and 'D': $cigar_2\n";
|
|
3055 }
|
|
3056 }
|
|
3057
|
|
3058 ### 3' end of read 2
|
|
3059 if ( ($methylation_call_params->{$sequence_identifier}->{index} == 0) or ($methylation_call_params->{$sequence_identifier}->{index} == 2) ){
|
|
3060 ## checking if the substring will be valid or if we can't extract the sequence because we are right at the edge of a chromosome
|
|
3061 unless (length($chromosomes{$methylation_call_params->{$sequence_identifier}->{chromosome}}) >= $pos_2+2){# exiting with en empty genomic sequence otherwise
|
|
3062 $methylation_call_params->{$sequence_identifier}->{unmodified_genomic_sequence_2} = $non_bisulfite_sequence_2;
|
|
3063 return;
|
|
3064 }
|
|
3065 $non_bisulfite_sequence_2 .= substr ($chromosomes{$methylation_call_params->{$sequence_identifier}->{chromosome}},$pos_2,2);
|
|
3066 }
|
|
3067
|
|
3068 ### all paired-end alignments reported by Bowtie 2 have the Read 1 alignment first and the Read 2 alignment as the second one irrespective of whether read 1 or read 2 was
|
|
3069 ### the + alignment. We also read in sequences read 1 then read 2 so they should correspond perfectly
|
|
3070
|
|
3071 ### results from CT converted read 1 plus GA converted read 2 vs. CT converted genome (+/- orientation alignments are reported only)
|
|
3072 if ($methylation_call_params->{$sequence_identifier}->{index} == 0){
|
|
3073 ### [Index 0, sequence originated from (converted) forward strand]
|
|
3074 $counting{CT_GA_CT_count}++;
|
|
3075 $alignment_read_1 = '+';
|
|
3076 $alignment_read_2 = '-';
|
|
3077 $read_conversion_info_1 = 'CT';
|
|
3078 $read_conversion_info_2 = 'GA';
|
|
3079 $genome_conversion = 'CT';
|
|
3080 ### Read 1 is always the forward hit
|
|
3081 ### Read 2 is will always on the reverse strand, so it needs to be reverse complemented
|
|
3082 $non_bisulfite_sequence_2 = reverse_complement($non_bisulfite_sequence_2);
|
|
3083 }
|
|
3084
|
|
3085 ### results from GA converted read 1 plus CT converted read 2 vs. GA converted genome (+/- orientation alignments are reported only)
|
|
3086 elsif ($methylation_call_params->{$sequence_identifier}->{index} == 1){
|
|
3087 ### [Index 1, sequence originated from complementary to (converted) bottom strand]
|
|
3088 $counting{GA_CT_GA_count}++;
|
|
3089 $alignment_read_1 = '+';
|
|
3090 $alignment_read_2 = '-';
|
|
3091 $read_conversion_info_1 = 'GA';
|
|
3092 $read_conversion_info_2 = 'CT';
|
|
3093 $genome_conversion = 'GA';
|
|
3094 ### Read 1 is always the forward hit
|
|
3095 ### Read 2 is will always on the reverse strand, so it needs to be reverse complemented
|
|
3096 $non_bisulfite_sequence_2 = reverse_complement($non_bisulfite_sequence_2);
|
|
3097 }
|
|
3098
|
|
3099 ### results from GA converted read 1 plus CT converted read 2 vs. CT converted genome (-/+ orientation alignments are reported only)
|
|
3100 elsif ($methylation_call_params->{$sequence_identifier}->{index} == 2){
|
|
3101 ### [Index 2, sequence originated from the complementary to (converted) top strand]
|
|
3102 $counting{GA_CT_CT_count}++;
|
|
3103 $alignment_read_1 = '-';
|
|
3104 $alignment_read_2 = '+';
|
|
3105 $read_conversion_info_1 = 'GA';
|
|
3106 $read_conversion_info_2 = 'CT';
|
|
3107 $genome_conversion = 'CT';
|
|
3108
|
|
3109 ### Read 1 (the reverse strand) genomic sequence needs to be reverse complemented
|
|
3110 $non_bisulfite_sequence_1 = reverse_complement($non_bisulfite_sequence_1);
|
|
3111 }
|
|
3112
|
|
3113 ### results from CT converted read 1 plus GA converted read 2 vs. GA converted genome (-/+ orientation alignments are reported only)
|
|
3114 elsif ($methylation_call_params->{$sequence_identifier}->{index} == 3){
|
|
3115 ### [Index 3, sequence originated from the (converted) reverse strand]
|
|
3116 $counting{CT_GA_GA_count}++;
|
|
3117 $alignment_read_1 = '-';
|
|
3118 $alignment_read_2 = '+';
|
|
3119 $read_conversion_info_1 = 'CT';
|
|
3120 $read_conversion_info_2 = 'GA';
|
|
3121 $genome_conversion = 'GA';
|
|
3122 ### Read 1 (the reverse strand) genomic sequence needs to be reverse complemented
|
|
3123 $non_bisulfite_sequence_1 = reverse_complement($non_bisulfite_sequence_1);
|
|
3124 }
|
|
3125 else{
|
|
3126 die "Too many bowtie result filehandles\n";
|
|
3127 }
|
|
3128 ### the alignment_strand information is needed to determine which strand of the genomic sequence we are comparing the read against,
|
|
3129 ### the read_conversion information is needed to know whether we are looking for C->T or G->A substitutions
|
|
3130
|
|
3131 $methylation_call_params->{$sequence_identifier}->{alignment_read_1} = $alignment_read_1;
|
|
3132 $methylation_call_params->{$sequence_identifier}->{alignment_read_2} = $alignment_read_2;
|
|
3133 $methylation_call_params->{$sequence_identifier}->{genome_conversion} = $genome_conversion;
|
|
3134 $methylation_call_params->{$sequence_identifier}->{read_conversion_1} = $read_conversion_info_1;
|
|
3135 $methylation_call_params->{$sequence_identifier}->{read_conversion_2} = $read_conversion_info_2;
|
|
3136 $methylation_call_params->{$sequence_identifier}->{unmodified_genomic_sequence_1} = $non_bisulfite_sequence_1;
|
|
3137 $methylation_call_params->{$sequence_identifier}->{unmodified_genomic_sequence_2} = $non_bisulfite_sequence_2;
|
|
3138 ## the end position of a read is stored in $pos
|
|
3139 $methylation_call_params->{$sequence_identifier}->{end_position_1} = $pos_1;
|
|
3140 $methylation_call_params->{$sequence_identifier}->{end_position_2} = $pos_2;
|
|
3141 $methylation_call_params->{$sequence_identifier}->{indels_1} = $indels_1;
|
|
3142 $methylation_call_params->{$sequence_identifier}->{indels_2} = $indels_2;
|
|
3143 }
|
|
3144
|
|
3145 ##########################################
|
|
3146 ### PRINT SINGLE END RESULTS: Bowtie 1 ###
|
|
3147 ##########################################
|
|
3148
|
|
3149 sub print_bisulfite_mapping_result_single_end{
|
|
3150 my ($identifier,$sequence,$methylation_call_params,$quality_value)= @_;
|
|
3151
|
|
3152 ### we will output the FastQ quality in Sanger encoding (Phred 33 scale)
|
|
3153 if ($phred64){
|
|
3154 $quality_value = convert_phred64_quals_to_phred33($quality_value);
|
|
3155 }
|
|
3156 elsif ($solexa){
|
|
3157 $quality_value = convert_solexa_quals_to_phred33($quality_value);
|
|
3158 }
|
|
3159
|
|
3160 ### We will add +1 bp to the starting position of single-end reads, as Bowtie 1 reports the index and not the bp position.
|
|
3161 $methylation_call_params->{$identifier}->{position} += 1;
|
|
3162
|
|
3163 ### writing every uniquely mapped read and its methylation call to the output file
|
|
3164 if ($vanilla){
|
|
3165 my $bowtie1_output = join("\t",$identifier,$methylation_call_params->{$identifier}->{alignment_strand},$methylation_call_params->{$identifier}->{chromosome},$methylation_call_params->{$identifier}->{position},$methylation_call_params->{$identifier}->{end_position},$sequence,$methylation_call_params->{$identifier}->{unmodified_genomic_sequence},$methylation_call_params->{$identifier}->{methylation_call},$methylation_call_params->{$identifier}->{read_conversion},$methylation_call_params->{$identifier}->{genome_conversion},$quality_value);
|
|
3166 print OUT "$bowtie1_output\n";
|
|
3167 }
|
|
3168 else{ # SAM output, default since Bismark v1.0.0
|
|
3169 single_end_SAM_output($identifier,$sequence,$methylation_call_params,$quality_value); # at the end of the script
|
|
3170 }
|
|
3171 }
|
|
3172
|
|
3173 ##########################################
|
|
3174 ### PRINT SINGLE END RESULTS: Bowtie 2 ###
|
|
3175 ##########################################
|
|
3176
|
|
3177 sub print_bisulfite_mapping_result_single_end_bowtie2{
|
|
3178 my ($identifier,$sequence,$methylation_call_params,$quality_value)= @_;
|
|
3179
|
|
3180 ### we will output the FastQ quality in Sanger encoding (Phred 33 scale)
|
|
3181 if ($phred64){
|
|
3182 $quality_value = convert_phred64_quals_to_phred33($quality_value);
|
|
3183 }
|
|
3184 elsif ($solexa){
|
|
3185 $quality_value = convert_solexa_quals_to_phred33($quality_value);
|
|
3186 }
|
|
3187
|
|
3188 ### writing every mapped read and its methylation call to the SAM output file (unmapped and ambiguous reads were already printed)
|
|
3189 single_end_SAM_output($identifier,$sequence,$methylation_call_params,$quality_value); # at the end of the script
|
|
3190 }
|
|
3191
|
|
3192 ##########################################
|
|
3193 ### PRINT PAIRED END ESULTS: Bowtie 1 ###
|
|
3194 ##########################################
|
|
3195
|
|
3196 sub print_bisulfite_mapping_results_paired_ends{
|
|
3197 my ($identifier,$sequence_1,$sequence_2,$methylation_call_params,$quality_value_1,$quality_value_2)= @_;
|
|
3198
|
|
3199 ### we will output the FastQ quality in Sanger encoding (Phred 33 scale)
|
|
3200 if ($phred64){
|
|
3201 $quality_value_1 = convert_phred64_quals_to_phred33($quality_value_1);
|
|
3202 $quality_value_2 = convert_phred64_quals_to_phred33($quality_value_2);
|
|
3203 }
|
|
3204 elsif ($solexa){
|
|
3205 $quality_value_1 = convert_solexa_quals_to_phred33($quality_value_1);
|
|
3206 $quality_value_2 = convert_solexa_quals_to_phred33($quality_value_2);
|
|
3207 }
|
|
3208
|
|
3209 ### We will add +1 bp to the start position of paired-end reads, as Bowtie 1 reports the index and not the bp position. (End position is already 1-based)
|
|
3210 $methylation_call_params->{$identifier}->{start_seq_1} += 1;
|
|
3211
|
|
3212 ### writing every single aligned read and its methylation call to the output file
|
|
3213 if ($vanilla){
|
|
3214 my $bowtie1_output_paired_end = join("\t",$identifier,$methylation_call_params->{$identifier}->{alignment_read_1},$methylation_call_params->{$identifier}->{chromosome},$methylation_call_params->{$identifier}->{start_seq_1},$methylation_call_params->{$identifier}->{alignment_end},$sequence_1,$methylation_call_params->{$identifier}->{unmodified_genomic_sequence_1},$methylation_call_params->{$identifier}->{methylation_call_1},$sequence_2,$methylation_call_params->{$identifier}->{unmodified_genomic_sequence_2},$methylation_call_params->{$identifier}->{methylation_call_2},$methylation_call_params->{$identifier}->{read_conversion_1},$methylation_call_params->{$identifier}->{genome_conversion},$quality_value_1,$quality_value_2);
|
|
3215 print OUT "$bowtie1_output_paired_end\n";
|
|
3216 }
|
|
3217 else{ # SAM output, default since Bismark v1.0.0
|
|
3218 paired_end_SAM_output($identifier,$sequence_1,$sequence_2,$methylation_call_params,$quality_value_1,$quality_value_2); # at the end of the script
|
|
3219 }
|
|
3220
|
|
3221 }
|
|
3222
|
|
3223 ##########################################
|
|
3224 ### PRINT PAIRED END ESULTS: Bowtie 2 ###
|
|
3225 ##########################################
|
|
3226
|
|
3227 sub print_bisulfite_mapping_results_paired_ends_bowtie2{
|
|
3228 my ($identifier,$sequence_1,$sequence_2,$methylation_call_params,$quality_value_1,$quality_value_2)= @_;
|
|
3229
|
|
3230 ### we will output the FastQ quality in Sanger encoding (Phred 33 scale)
|
|
3231 if ($phred64){
|
|
3232 $quality_value_1 = convert_phred64_quals_to_phred33($quality_value_1);
|
|
3233 $quality_value_2 = convert_phred64_quals_to_phred33($quality_value_2);
|
|
3234 }
|
|
3235 elsif ($solexa){
|
|
3236 $quality_value_1 = convert_solexa_quals_to_phred33($quality_value_1);
|
|
3237 $quality_value_2 = convert_solexa_quals_to_phred33($quality_value_2);
|
|
3238 }
|
|
3239
|
|
3240 ### writing every single aligned read and its methylation call to the output file (unmapped and ambiguous reads were already printed)
|
|
3241 paired_end_SAM_output($identifier,$sequence_1,$sequence_2,$methylation_call_params,$quality_value_1,$quality_value_2); # at the end of the script
|
|
3242
|
|
3243 }
|
|
3244
|
|
3245
|
|
3246 sub convert_phred64_quals_to_phred33{
|
|
3247
|
|
3248 my $qual = shift;
|
|
3249 my @quals = split (//,$qual);
|
|
3250 my @new_quals;
|
|
3251
|
|
3252 foreach my $index (0..$#quals){
|
|
3253 my $phred_score = convert_phred64_quality_string_into_phred_score ($quals[$index]);
|
|
3254 my $phred33_quality_string = convert_phred_score_into_phred33_quality_string ($phred_score);
|
|
3255 $new_quals[$index] = $phred33_quality_string;
|
|
3256 }
|
|
3257
|
|
3258 my $phred33_quality = join ("",@new_quals);
|
|
3259 return $phred33_quality;
|
|
3260 }
|
|
3261
|
|
3262 sub convert_solexa_quals_to_phred33{
|
|
3263
|
|
3264 my $qual = shift;
|
|
3265 my @quals = split (//,$qual);
|
|
3266 my @new_quals;
|
|
3267
|
|
3268 foreach my $index (0..$#quals){
|
|
3269 my $phred_score = convert_solexa_pre1_3_quality_string_into_phred_score ($quals[$index]);
|
|
3270 my $phred33_quality_string = convert_phred_score_into_phred33_quality_string ($phred_score);
|
|
3271 $new_quals[$index] = $phred33_quality_string;
|
|
3272 }
|
|
3273
|
|
3274 my $phred33_quality = join ("",@new_quals);
|
|
3275 return $phred33_quality;
|
|
3276 }
|
|
3277
|
|
3278 sub convert_phred_score_into_phred33_quality_string{
|
|
3279 my $qual = shift;
|
|
3280 $qual = chr($qual+33);
|
|
3281 return $qual;
|
|
3282 }
|
|
3283
|
|
3284 sub convert_phred64_quality_string_into_phred_score{
|
|
3285 my $string = shift;
|
|
3286 my $qual = ord($string)-64;
|
|
3287 return $qual;
|
|
3288 }
|
|
3289
|
|
3290 sub convert_solexa_pre1_3_quality_string_into_phred_score{
|
|
3291 ### We will just use 59 as the offset here as all Phred Scores between 10 and 40 look exactly the same, there is only a minute difference for values between 0 and 10
|
|
3292 my $string = shift;
|
|
3293 my $qual = ord($string)-59;
|
|
3294 return $qual;
|
|
3295 }
|
|
3296
|
|
3297
|
|
3298 sub extract_corresponding_genomic_sequence_single_end {
|
|
3299 my ($sequence_identifier,$methylation_call_params) = @_;
|
|
3300 ### A bisulfite sequence for 1 location in the genome can theoretically be any of the 4 possible converted strands. We are also giving the
|
|
3301 ### sequence a 'memory' of the conversion we are expecting which we will need later for the methylation call
|
|
3302
|
|
3303 ### the alignment_strand information is needed to determine which strand of the genomic sequence we are comparing the read against,
|
|
3304 ### the read_conversion information is needed to know whether we are looking for C->T or G->A substitutions
|
|
3305 my $alignment_strand;
|
|
3306 my $read_conversion_info;
|
|
3307 my $genome_conversion;
|
|
3308 ### Also extracting the corresponding genomic sequence, +2 extra bases at the end so that we can also make a CpG methylation call and
|
|
3309 ### in addition make differential calls for Cs non-CpG context, which will now be divided into CHG and CHH methylation,
|
|
3310 ### if the C happens to be at the last position of the actually observed sequence
|
|
3311 my $non_bisulfite_sequence;
|
|
3312 ### depending on the conversion we want to make need to capture 1 extra base at the 3' end
|
|
3313
|
|
3314 ### results from CT converted read vs. CT converted genome (+ orientation alignments are reported only)
|
|
3315 if ($methylation_call_params->{$sequence_identifier}->{index} == 0){
|
|
3316 ### [Index 0, sequence originated from (converted) forward strand]
|
|
3317 $counting{CT_CT_count}++;
|
|
3318 $alignment_strand = '+';
|
|
3319 $read_conversion_info = 'CT';
|
|
3320 $genome_conversion = 'CT';
|
|
3321
|
|
3322 ## checking if the substring will be valid or if we can't extract the sequence because we are right at the edge of a chromosome
|
|
3323 if (length($chromosomes{$methylation_call_params->{$sequence_identifier}->{chromosome}}) > $methylation_call_params->{$sequence_identifier}->{position}+length($methylation_call_params->{$sequence_identifier}->{bowtie_sequence})+1){ ## CHH changed to +1
|
|
3324 ### + 2 extra base at the 3' end
|
|
3325 $non_bisulfite_sequence = substr ($chromosomes{$methylation_call_params->{$sequence_identifier}->{chromosome}},$methylation_call_params->{$sequence_identifier}->{position},length($methylation_call_params->{$sequence_identifier}->{bowtie_sequence})+2); ## CHH changed to +2
|
|
3326 }
|
|
3327 else{
|
|
3328 $non_bisulfite_sequence = '';
|
|
3329 }
|
|
3330 }
|
|
3331
|
|
3332 ### results from CT converted reads vs. GA converted genome (- orientation alignments are reported only)
|
|
3333 elsif ($methylation_call_params->{$sequence_identifier}->{index} == 1){
|
|
3334 ### [Index 1, sequence originated from (converted) reverse strand]
|
|
3335 $counting{CT_GA_count}++;
|
|
3336 $alignment_strand = '-';
|
|
3337 $read_conversion_info = 'CT';
|
|
3338 $genome_conversion = 'GA';
|
|
3339
|
|
3340 ## checking if the substring will be valid or if we can't extract the sequence because we are right at the edge of a chromosome
|
|
3341 if ($methylation_call_params->{$sequence_identifier}->{position}-2 >= 0){ ## CHH changed to -2 # 02 02 2012 Changed this to >= from >
|
|
3342 ### Extracting 2 extra 5' bases on forward strand which will become 2 extra 3' bases after reverse complementation
|
|
3343 $non_bisulfite_sequence = substr ($chromosomes{$methylation_call_params->{$sequence_identifier}->{chromosome}},$methylation_call_params->{$sequence_identifier}->{position}-2,length($methylation_call_params->{$sequence_identifier}->{bowtie_sequence})+2); ## CHH changed to -2/+2
|
|
3344 ## reverse complement!
|
|
3345 $non_bisulfite_sequence = reverse_complement($non_bisulfite_sequence);
|
|
3346 }
|
|
3347 else{
|
|
3348 $non_bisulfite_sequence = '';
|
|
3349 }
|
|
3350 }
|
|
3351
|
|
3352 ### results from GA converted reads vs. CT converted genome (- orientation alignments are reported only)
|
|
3353 elsif ($methylation_call_params->{$sequence_identifier}->{index} == 2){
|
|
3354 ### [Index 2, sequence originated from complementary to (converted) forward strand]
|
|
3355 $counting{GA_CT_count}++;
|
|
3356 $alignment_strand = '-';
|
|
3357 $read_conversion_info = 'GA';
|
|
3358 $genome_conversion = 'CT';
|
|
3359
|
|
3360 ### +2 extra bases on the forward strand 3', which will become 2 extra 5' bases after reverse complementation
|
|
3361 ## checking if the substring will be valid or if we can't extract the sequence because we are right at the edge of a chromosome
|
|
3362 if (length($chromosomes{$methylation_call_params->{$sequence_identifier}->{chromosome}}) > $methylation_call_params->{$sequence_identifier}->{position}+length($methylation_call_params->{$sequence_identifier}->{bowtie_sequence})+1){ ## changed to +1 on 02 02 2012
|
|
3363 $non_bisulfite_sequence = substr ($chromosomes{$methylation_call_params->{$sequence_identifier}->{chromosome}},$methylation_call_params->{$sequence_identifier}->{position},length($methylation_call_params->{$sequence_identifier}->{bowtie_sequence})+2); ## CHH changed to +2
|
|
3364 ## reverse complement!
|
|
3365 $non_bisulfite_sequence = reverse_complement($non_bisulfite_sequence);
|
|
3366 }
|
|
3367 else{
|
|
3368 $non_bisulfite_sequence = '';
|
|
3369 }
|
|
3370 }
|
|
3371
|
|
3372 ### results from GA converted reads vs. GA converted genome (+ orientation alignments are reported only)
|
|
3373 elsif ($methylation_call_params->{$sequence_identifier}->{index} == 3){
|
|
3374 ### [Index 3, sequence originated from complementary to (converted) reverse strand]
|
|
3375 $counting{GA_GA_count}++;
|
|
3376 $alignment_strand = '+';
|
|
3377 $read_conversion_info = 'GA';
|
|
3378 $genome_conversion = 'GA';
|
|
3379
|
|
3380 ## checking if the substring will be valid or if we can't extract the sequence because we are right at the edge of a chromosome
|
|
3381 if ($methylation_call_params->{$sequence_identifier}->{position}-2 >= 0){ ## CHH changed to +2 # 02 02 2012 Changed this to >= from >
|
|
3382 ### +2 extra base at the 5' end as we are nominally checking the converted reverse strand
|
|
3383 $non_bisulfite_sequence = substr ($chromosomes{$methylation_call_params->{$sequence_identifier}->{chromosome}},$methylation_call_params->{$sequence_identifier}->{position}-2,length($methylation_call_params->{$sequence_identifier}->{bowtie_sequence})+2); ## CHH changed to -2/+2
|
|
3384 }
|
|
3385 else{
|
|
3386 $non_bisulfite_sequence = '';
|
|
3387 }
|
|
3388 }
|
|
3389 else{
|
|
3390 die "Too many bowtie result filehandles\n";
|
|
3391 }
|
|
3392
|
|
3393 $methylation_call_params->{$sequence_identifier}->{alignment_strand} = $alignment_strand;
|
|
3394 $methylation_call_params->{$sequence_identifier}->{read_conversion} = $read_conversion_info;
|
|
3395 $methylation_call_params->{$sequence_identifier}->{genome_conversion} = $genome_conversion;
|
|
3396 $methylation_call_params->{$sequence_identifier}->{unmodified_genomic_sequence} = $non_bisulfite_sequence;
|
|
3397
|
|
3398 ### at this point we can also determine the end position of a read
|
|
3399 $methylation_call_params->{$sequence_identifier}->{end_position} = $methylation_call_params->{$sequence_identifier}->{position}+length($methylation_call_params->{$sequence_identifier}->{bowtie_sequence});
|
|
3400 }
|
|
3401
|
|
3402
|
|
3403 sub extract_corresponding_genomic_sequence_single_end_bowtie2{
|
|
3404 my ($sequence_identifier,$methylation_call_params) = @_;
|
|
3405
|
|
3406 my $MD_tag = $methylation_call_params->{$sequence_identifier}->{mismatch_info};
|
|
3407 my $cigar = $methylation_call_params->{$sequence_identifier}->{CIGAR};
|
|
3408
|
|
3409 ### A bisulfite sequence for 1 location in the genome can theoretically be any of the 4 possible converted strands. We are also giving the
|
|
3410 ### sequence a 'memory' of the conversion we are expecting which we will need later for the methylation call
|
|
3411
|
|
3412 ### the alignment_strand information is needed to determine which strand of the genomic sequence we are comparing the read against,
|
|
3413 ### the read_conversion information is needed to know whether we are looking for C->T or G->A substitutions
|
|
3414 my $alignment_strand;
|
|
3415 my $read_conversion_info;
|
|
3416 my $genome_conversion;
|
|
3417 ### We are now extracting the corresponding genomic sequence, +2 extra bases at the end (or start) so that we can also make a CpG methylation call and
|
|
3418 ### in addition make differential calls for Cs in CHG or CHH context if the C happens to be at the last (or first) position of the actually observed sequence
|
|
3419 my $non_bisulfite_sequence = '';
|
|
3420
|
|
3421 ### Positions in SAM format are 1 based, so we need to subract 1 when getting substrings
|
|
3422 my $pos = $methylation_call_params->{$sequence_identifier}->{position}-1;
|
|
3423
|
|
3424 # parsing CIGAR string
|
|
3425 my @len = split (/\D+/,$cigar); # storing the length per operation
|
|
3426 my @ops = split (/\d+/,$cigar); # storing the operation
|
|
3427 shift @ops; # remove the empty first element
|
|
3428 die "CIGAR string contained a non-matching number of lengths and operations\n" unless (scalar @len == scalar @ops);
|
|
3429
|
|
3430 ### If the sequence aligns best as CT converted reads vs. GA converted genome (OB, index 1) or GA converted reads vs. GA converted genome (CTOB, index 3)
|
|
3431 if ( ($methylation_call_params->{$sequence_identifier}->{index} == 1) or ($methylation_call_params->{$sequence_identifier}->{index} == 3) ){
|
|
3432 ## checking if the substring will be valid or if we can't extract the sequence because we are right at the edge of a chromosome
|
|
3433 unless ( ($pos-2) >= 0){ # exiting with en empty genomic sequence otherwise
|
|
3434 $methylation_call_params->{$sequence_identifier}->{unmodified_genomic_sequence} = $non_bisulfite_sequence;
|
|
3435 return;
|
|
3436 }
|
|
3437 $non_bisulfite_sequence .= substr ($chromosomes{$methylation_call_params->{$sequence_identifier}->{chromosome}},$pos-2,2);
|
|
3438 }
|
|
3439 my $indels = 0;
|
|
3440
|
|
3441 foreach (0..$#len){
|
|
3442 if ($ops[$_] eq 'M'){
|
|
3443 #extracting genomic sequence
|
|
3444 $non_bisulfite_sequence .= substr ($chromosomes{$methylation_call_params->{$sequence_identifier}->{chromosome}},$pos,$len[$_]);
|
|
3445 # adjusting position
|
|
3446 $pos += $len[$_];
|
|
3447 }
|
|
3448 elsif ($ops[$_] eq 'I'){ # insertion in the read sequence
|
|
3449 # we simply add padding Ns instead of finding genomic sequence. This will not be used to infer methylation calls
|
|
3450 $non_bisulfite_sequence .= 'N' x $len[$_];
|
|
3451 # warn "$non_bisulfite_sequence\n";
|
|
3452 # position doesn't need to be adjusting
|
|
3453 $indels += $len[$_]; # adding this to $indels so we can determine the hemming distance for the SAM output (= single-base substitutions (mismatches, insertions, deletions)
|
|
3454 }
|
|
3455 elsif ($ops[$_] eq 'D'){ # deletion in the read sequence
|
|
3456 # we do not add any genomic sequence but only adjust the position
|
|
3457 $pos += $len[$_];
|
|
3458 $indels += $len[$_]; # adding this to $indels so we can determine the hemming distance for the SAM output (= single-base substitutions (mismatches, insertions, deletions)
|
|
3459 }
|
|
3460 elsif($cigar =~ tr/[NSHPX=]//){ # if these (for standard mapping) illegal characters exist we die
|
|
3461 die "The CIGAR string contained illegal CIGAR operations in addition to 'M', 'I' and 'D': $cigar\n";
|
|
3462 }
|
|
3463 else{
|
|
3464 die "The CIGAR string contained undefined CIGAR operations in addition to 'M', 'I' and 'D': $cigar\n";
|
|
3465 }
|
|
3466 }
|
|
3467
|
|
3468 ### If the sequence aligns best as CT converted reads vs. CT converted genome (OT, index 0) or GA converted reads vs. CT converted genome (CTOT, index 2)
|
|
3469 if ( ($methylation_call_params->{$sequence_identifier}->{index} == 0) or ($methylation_call_params->{$sequence_identifier}->{index} == 2) ){
|
|
3470 ## checking if the substring will be valid or if we can't extract the sequence because we are right at the edge of a chromosome
|
|
3471 unless (length($chromosomes{$methylation_call_params->{$sequence_identifier}->{chromosome}}) >= $pos+2){ # exiting with en empty genomic sequence otherwise
|
|
3472 $methylation_call_params->{$sequence_identifier}->{unmodified_genomic_sequence} = $non_bisulfite_sequence;
|
|
3473 return;
|
|
3474 }
|
|
3475 $non_bisulfite_sequence .= substr ($chromosomes{$methylation_call_params->{$sequence_identifier}->{chromosome}},$pos,2);
|
|
3476 # print "$methylation_call_params->{$sequence_identifier}->{bowtie_sequence}\n$non_bisulfite_sequence\n";
|
|
3477 }
|
|
3478
|
|
3479
|
|
3480
|
|
3481 ### results from CT converted read vs. CT converted genome (+ orientation alignments are reported only)
|
|
3482 if ($methylation_call_params->{$sequence_identifier}->{index} == 0){
|
|
3483 ### [Index 0, sequence originated from (converted) forward strand]
|
|
3484 $counting{CT_CT_count}++;
|
|
3485 $alignment_strand = '+';
|
|
3486 $read_conversion_info = 'CT';
|
|
3487 $genome_conversion = 'CT';
|
|
3488 }
|
|
3489
|
|
3490 ### results from CT converted reads vs. GA converted genome (- orientation alignments are reported only)
|
|
3491 elsif ($methylation_call_params->{$sequence_identifier}->{index} == 1){
|
|
3492 ### [Index 1, sequence originated from (converted) reverse strand]
|
|
3493 $counting{CT_GA_count}++;
|
|
3494 $alignment_strand = '-';
|
|
3495 $read_conversion_info = 'CT';
|
|
3496 $genome_conversion = 'GA';
|
|
3497
|
|
3498 ### reverse complement!
|
|
3499 $non_bisulfite_sequence = reverse_complement($non_bisulfite_sequence);
|
|
3500 }
|
|
3501
|
|
3502 ### results from GA converted reads vs. CT converted genome (- orientation alignments are reported only)
|
|
3503 elsif ($methylation_call_params->{$sequence_identifier}->{index} == 2){
|
|
3504 ### [Index 2, sequence originated from complementary to (converted) forward strand]
|
|
3505 $counting{GA_CT_count}++;
|
|
3506 $alignment_strand = '-';
|
|
3507 $read_conversion_info = 'GA';
|
|
3508 $genome_conversion = 'CT';
|
|
3509
|
|
3510 ### reverse complement!
|
|
3511 $non_bisulfite_sequence = reverse_complement($non_bisulfite_sequence);
|
|
3512 }
|
|
3513
|
|
3514 ### results from GA converted reads vs. GA converted genome (+ orientation alignments are reported only)
|
|
3515 elsif ($methylation_call_params->{$sequence_identifier}->{index} == 3){
|
|
3516 ### [Index 3, sequence originated from complementary to (converted) reverse strand]
|
|
3517 $counting{GA_GA_count}++;
|
|
3518 $alignment_strand = '+';
|
|
3519 $read_conversion_info = 'GA';
|
|
3520 $genome_conversion = 'GA';
|
|
3521
|
|
3522 }
|
|
3523 else{
|
|
3524 die "Too many Bowtie 2 result filehandles\n";
|
|
3525 }
|
|
3526
|
|
3527 $methylation_call_params->{$sequence_identifier}->{alignment_strand} = $alignment_strand;
|
|
3528 $methylation_call_params->{$sequence_identifier}->{read_conversion} = $read_conversion_info;
|
|
3529 $methylation_call_params->{$sequence_identifier}->{genome_conversion} = $genome_conversion;
|
|
3530 $methylation_call_params->{$sequence_identifier}->{unmodified_genomic_sequence} = $non_bisulfite_sequence;
|
|
3531
|
|
3532 ### the end position of a read is stored in $pos
|
|
3533 $methylation_call_params->{$sequence_identifier}->{end_position} = $pos;
|
|
3534 $methylation_call_params->{$sequence_identifier}->{indels} = $indels;
|
|
3535 }
|
|
3536
|
|
3537 ### METHYLATION CALL
|
|
3538
|
|
3539 sub methylation_call{
|
|
3540 my ($identifier,$sequence_actually_observed,$genomic_sequence,$read_conversion) = @_;
|
|
3541 ### splitting both the actually observed sequence and the genomic sequence up into single bases so we can compare them one by one
|
|
3542 my @seq = split(//,$sequence_actually_observed);
|
|
3543 my @genomic = split(//,$genomic_sequence);
|
|
3544 # print join ("\n",$identifier,$sequence_actually_observed,$genomic_sequence,$read_conversion),"\n";
|
|
3545 ### Creating a match-string with different characters for non-cytosine bases (disregarding mismatches here), methyl-Cs or non-methyl Cs in either
|
|
3546 ### CpG, CHH or CHG context
|
|
3547
|
|
3548 #################################################################
|
|
3549 ### . for bases not involving cytosines ###
|
|
3550 ### X for methylated C in CHG context (was protected) ###
|
|
3551 ### x for not methylated C in CHG context (was converted) ###
|
|
3552 ### H for methylated C in CHH context (was protected) ###
|
|
3553 ### h for not methylated C in CHH context (was converted) ###
|
|
3554 ### Z for methylated C in CpG context (was protected) ###
|
|
3555 ### z for not methylated C in CpG context (was converted) ###
|
|
3556 #################################################################
|
|
3557
|
|
3558 my @match =();
|
|
3559 warn "length of \@seq: ",scalar @seq,"\tlength of \@genomic: ",scalar @genomic,"\n" unless (scalar @seq eq (scalar@genomic-2)); ## CHH changed to -2
|
|
3560 my $methyl_CHH_count = 0;
|
|
3561 my $methyl_CHG_count = 0;
|
|
3562 my $methyl_CpG_count = 0;
|
|
3563 my $unmethylated_CHH_count = 0;
|
|
3564 my $unmethylated_CHG_count = 0;
|
|
3565 my $unmethylated_CpG_count = 0;
|
|
3566
|
|
3567 if ($read_conversion eq 'CT'){
|
|
3568 for my $index (0..$#seq) {
|
|
3569 if ($seq[$index] eq $genomic[$index]) {
|
|
3570 ### The residue can only be a C if it was not converted to T, i.e. protected my methylation
|
|
3571 if ($genomic[$index] eq 'C') {
|
|
3572 ### If the residue is a C we want to know if it was in CpG context or in any other context
|
|
3573 my $downstream_base = $genomic[$index+1];
|
|
3574
|
|
3575 if ($downstream_base eq 'G'){
|
|
3576 ++$methyl_CpG_count;
|
|
3577 push @match,'Z'; # protected C, methylated, in CpG context
|
|
3578 }
|
|
3579
|
|
3580 else {
|
|
3581 ### C in not in CpG-context, determining the second downstream base context
|
|
3582 my $second_downstream_base = $genomic[$index+2];
|
|
3583
|
|
3584 if ($second_downstream_base eq 'G'){
|
|
3585 ++$methyl_CHG_count;
|
|
3586 push @match,'X'; # protected C, methylated, in CHG context
|
|
3587 }
|
|
3588 else{
|
|
3589 ++$methyl_CHH_count;
|
|
3590 push @match,'H'; # protected C, methylated, in CHH context
|
|
3591 }
|
|
3592 }
|
|
3593 }
|
|
3594 else {
|
|
3595 push @match, '.';
|
|
3596 }
|
|
3597 }
|
|
3598 elsif ($seq[$index] ne $genomic[$index]) {
|
|
3599 ### for the methylation call we are only interested in mismatches involving cytosines (in the genomic sequence) which were converted into Ts
|
|
3600 ### in the actually observed sequence
|
|
3601 if ($genomic[$index] eq 'C' and $seq[$index] eq 'T') {
|
|
3602 ### If the residue was converted to T we want to know if it was in CpG, CHG or CHH context
|
|
3603 my $downstream_base = $genomic[$index+1];
|
|
3604
|
|
3605 if ($downstream_base eq 'G'){
|
|
3606 ++$unmethylated_CpG_count;
|
|
3607 push @match,'z'; # converted C, not methylated, in CpG context
|
|
3608 }
|
|
3609
|
|
3610 else{
|
|
3611 ### C in not in CpG-context, determining the second downstream base context
|
|
3612 my $second_downstream_base = $genomic[$index+2];
|
|
3613
|
|
3614 if ($second_downstream_base eq 'G'){
|
|
3615 ++$unmethylated_CHG_count;
|
|
3616 push @match,'x'; # converted C, not methylated, in CHG context
|
|
3617 }
|
|
3618 else{
|
|
3619 ++$unmethylated_CHH_count;
|
|
3620 push @match,'h'; # converted C, not methylated, in CHH context
|
|
3621 }
|
|
3622 }
|
|
3623 }
|
|
3624 ### all other mismatches are not of interest for a methylation call
|
|
3625 else {
|
|
3626 push @match,'.';
|
|
3627 }
|
|
3628 }
|
|
3629 else{
|
|
3630 die "There can be only 2 possibilities\n";
|
|
3631 }
|
|
3632 }
|
|
3633 }
|
|
3634 elsif ($read_conversion eq 'GA'){
|
|
3635 # print join ("\n",'***',$identifier,$sequence_actually_observed,$genomic_sequence,$read_conversion,'***'),"\n";
|
|
3636
|
|
3637 for my $index (0..$#seq) {
|
|
3638 if ($seq[$index] eq $genomic[$index+2]) {
|
|
3639 ### The residue can only be a G if the C on the other strand was not converted to T, i.e. protected my methylation
|
|
3640 if ($genomic[$index+2] eq 'G') {
|
|
3641 ### If the residue is a G we want to know if the C on the other strand was in CpG, CHG or CHH context, therefore we need
|
|
3642 ### to look if the base upstream is a C
|
|
3643
|
|
3644 my $upstream_base = $genomic[$index+1];
|
|
3645
|
|
3646 if ($upstream_base eq 'C'){
|
|
3647 ++$methyl_CpG_count;
|
|
3648 push @match,'Z'; # protected C on opposing strand, methylated, in CpG context
|
|
3649 }
|
|
3650
|
|
3651 else{
|
|
3652 ### C in not in CpG-context, determining the second upstream base context
|
|
3653 my $second_upstream_base = $genomic[$index];
|
|
3654
|
|
3655 if ($second_upstream_base eq 'C'){
|
|
3656 ++$methyl_CHG_count;
|
|
3657 push @match,'X'; # protected C on opposing strand, methylated, in CHG context
|
|
3658 }
|
|
3659 else{
|
|
3660 ++$methyl_CHH_count;
|
|
3661 push @match,'H'; # protected C on opposing strand, methylated, in CHH context
|
|
3662 }
|
|
3663 }
|
|
3664 }
|
|
3665 else{
|
|
3666 push @match, '.';
|
|
3667 }
|
|
3668 }
|
|
3669 elsif ($seq[$index] ne $genomic[$index+2]) {
|
|
3670 ### for the methylation call we are only interested in mismatches involving cytosines (in the genomic sequence) which were converted to Ts
|
|
3671 ### on the opposing strand, so G to A conversions in the actually observed sequence
|
|
3672 if ($genomic[$index+2] eq 'G' and $seq[$index] eq 'A') {
|
|
3673 ### If the C residue on the opposing strand was converted to T then we will see an A in the currently observed sequence. We want to know if
|
|
3674 ### the C on the opposing strand was it was in CpG, CHG or CHH context, therefore we need to look one (or two) bases upstream!
|
|
3675
|
|
3676 my $upstream_base = $genomic[$index+1];
|
|
3677
|
|
3678 if ($upstream_base eq 'C'){
|
|
3679 ++$unmethylated_CpG_count;
|
|
3680 push @match,'z'; # converted C on opposing strand, not methylated, in CpG context
|
|
3681 }
|
|
3682
|
|
3683 else{
|
|
3684 ### C in not in CpG-context, determining the second upstream base context
|
|
3685 my $second_upstream_base = $genomic[$index];
|
|
3686
|
|
3687 if ($second_upstream_base eq 'C'){
|
|
3688 ++$unmethylated_CHG_count;
|
|
3689 push @match,'x'; # converted C on opposing strand, not methylated, in CHG context
|
|
3690 }
|
|
3691 else{
|
|
3692 ++$unmethylated_CHH_count;
|
|
3693 push @match,'h'; # converted C on opposing strand, not methylated, in CHH context
|
|
3694 }
|
|
3695 }
|
|
3696 }
|
|
3697 ### all other mismatches are not of interest for a methylation call
|
|
3698 else {
|
|
3699 push @match,'.';
|
|
3700 }
|
|
3701 }
|
|
3702 else{
|
|
3703 die "There can be only 2 possibilities\n";
|
|
3704 }
|
|
3705 }
|
|
3706 }
|
|
3707 else{
|
|
3708 die "Strand conversion info is required to perform a methylation call\n";
|
|
3709 }
|
|
3710
|
|
3711 my $methylation_call = join ("",@match);
|
|
3712
|
|
3713 $counting{total_meCHH_count} += $methyl_CHH_count;
|
|
3714 $counting{total_meCHG_count} += $methyl_CHG_count;
|
|
3715 $counting{total_meCpG_count} += $methyl_CpG_count;
|
|
3716 $counting{total_unmethylated_CHH_count} += $unmethylated_CHH_count;
|
|
3717 $counting{total_unmethylated_CHG_count} += $unmethylated_CHG_count;
|
|
3718 $counting{total_unmethylated_CpG_count} += $unmethylated_CpG_count;
|
|
3719
|
|
3720 # print "\n$sequence_actually_observed\n$genomic_sequence\n",@match,"\n$read_conversion\n\n";
|
|
3721 return $methylation_call;
|
|
3722 }
|
|
3723
|
|
3724 sub read_genome_into_memory{
|
|
3725 ## working directoy
|
|
3726 my $cwd = shift;
|
|
3727 ## reading in and storing the specified genome in the %chromosomes hash
|
|
3728 chdir ($genome_folder) or die "Can't move to $genome_folder: $!";
|
|
3729 print "Now reading in and storing sequence information of the genome specified in: $genome_folder\n\n";
|
|
3730
|
|
3731 my @chromosome_filenames = <*.fa>;
|
|
3732
|
|
3733 ### if there aren't any genomic files with the extension .fa we will look for files with the extension .fasta
|
|
3734 unless (@chromosome_filenames){
|
|
3735 @chromosome_filenames = <*.fasta>;
|
|
3736 }
|
|
3737
|
|
3738 unless (@chromosome_filenames){
|
|
3739 die "The specified genome folder $genome_folder does not contain any sequence files in FastA format (with .fa or .fasta file extensions)\n";
|
|
3740 }
|
|
3741
|
|
3742 foreach my $chromosome_filename (@chromosome_filenames){
|
|
3743
|
|
3744 open (CHR_IN,$chromosome_filename) or die "Failed to read from sequence file $chromosome_filename $!\n";
|
|
3745 ### first line needs to be a fastA header
|
|
3746 my $first_line = <CHR_IN>;
|
|
3747 chomp $first_line;
|
|
3748 $first_line =~ s/\r//;
|
|
3749
|
|
3750 ### Extracting chromosome name from the FastA header
|
|
3751 my $chromosome_name = extract_chromosome_name($first_line);
|
|
3752
|
|
3753 my $sequence;
|
|
3754 while (<CHR_IN>){
|
|
3755 chomp;
|
|
3756 $_ =~ s/\r//;
|
|
3757 if ($_ =~ /^>/){
|
|
3758 ### storing the previous chromosome in the %chromosomes hash, only relevant for Multi-Fasta-Files (MFA)
|
|
3759 if (exists $chromosomes{$chromosome_name}){
|
|
3760 print "chr $chromosome_name (",length $sequence ," bp)\n";
|
|
3761 die "Exiting because chromosome name already exists. Please make sure all chromosomes have a unique name!\n";
|
|
3762 }
|
|
3763 else {
|
|
3764 if (length($sequence) == 0){
|
|
3765 warn "Chromosome $chromosome_name in the multi-fasta file $chromosome_filename did not contain any sequence information!\n";
|
|
3766 }
|
|
3767 print "chr $chromosome_name (",length $sequence ," bp)\n";
|
|
3768 $chromosomes{$chromosome_name} = $sequence;
|
|
3769 }
|
|
3770 ### resetting the sequence variable
|
|
3771 $sequence = '';
|
|
3772 ### setting new chromosome name
|
|
3773 $chromosome_name = extract_chromosome_name($_);
|
|
3774 }
|
|
3775 else{
|
|
3776 $sequence .= uc$_;
|
|
3777 }
|
|
3778 }
|
|
3779
|
|
3780 if (exists $chromosomes{$chromosome_name}){
|
|
3781 print "chr $chromosome_name (",length $sequence ," bp)\t";
|
|
3782 die "Exiting because chromosome name already exists. Please make sure all chromosomes have a unique name.\n";
|
|
3783 }
|
|
3784 else{
|
|
3785 if (length($sequence) == 0){
|
|
3786 warn "Chromosome $chromosome_name in the file $chromosome_filename did not contain any sequence information!\n";
|
|
3787 }
|
|
3788 print "chr $chromosome_name (",length $sequence ," bp)\n";
|
|
3789 $chromosomes{$chromosome_name} = $sequence;
|
|
3790 }
|
|
3791 }
|
|
3792 print "\n";
|
|
3793 chdir $cwd or die "Failed to move to directory $cwd\n";
|
|
3794 }
|
|
3795
|
|
3796 sub extract_chromosome_name {
|
|
3797 ## Bowtie seems to extract the first string after the inition > in the FASTA file, so we are doing this as well
|
|
3798 my $fasta_header = shift;
|
|
3799 if ($fasta_header =~ s/^>//){
|
|
3800 my ($chromosome_name) = split (/\s+/,$fasta_header);
|
|
3801 return $chromosome_name;
|
|
3802 }
|
|
3803 else{
|
|
3804 die "The specified chromosome ($fasta_header) file doesn't seem to be in FASTA format as required!\n";
|
|
3805 }
|
|
3806 }
|
|
3807
|
|
3808 sub reverse_complement{
|
|
3809 my $sequence = shift;
|
|
3810 $sequence =~ tr/CATG/GTAC/;
|
|
3811 $sequence = reverse($sequence);
|
|
3812 return $sequence;
|
|
3813 }
|
|
3814
|
|
3815 sub biTransformFastAFiles {
|
|
3816 my $file = shift;
|
|
3817 my ($dir,$filename);
|
|
3818 if ($file =~ /\//){
|
|
3819 ($dir,$filename) = $file =~ m/(.*\/)(.*)$/;
|
|
3820 }
|
|
3821 else{
|
|
3822 $filename = $file;
|
|
3823 }
|
|
3824
|
|
3825 ### gzipped version of the infile
|
|
3826 if ($file =~ /\.gz$/){
|
|
3827 open (IN,"zcat $file |") or die "Couldn't read from file $file: $!\n";
|
|
3828 }
|
|
3829 else{
|
|
3830 open (IN,$file) or die "Couldn't read from file $file: $!\n";
|
|
3831 }
|
|
3832
|
|
3833 if ($skip){
|
|
3834 warn "Skipping the first $skip reads from $file\n";
|
|
3835 sleep (1);
|
|
3836 }
|
|
3837 if ($upto){
|
|
3838 warn "Processing reads up to sequence no. $upto from $file\n";
|
|
3839 sleep (1);
|
|
3840 }
|
|
3841
|
|
3842 my $C_to_T_infile = my $G_to_A_infile = $filename;
|
|
3843 $C_to_T_infile =~ s/$/_C_to_T.fa/;
|
|
3844 $G_to_A_infile =~ s/$/_G_to_A.fa/;
|
|
3845 print "Writing a C -> T converted version of the input file $filename to $temp_dir$C_to_T_infile\n";
|
|
3846 open (CTOT,'>',"$temp_dir$C_to_T_infile") or die "Couldn't write to file $!\n";
|
|
3847
|
|
3848 unless ($directional){
|
|
3849 print "Writing a G -> A converted version of the input file $filename to $temp_dir$G_to_A_infile\n";
|
|
3850 open (GTOA,'>',"$temp_dir$G_to_A_infile") or die "Couldn't write to file $!\n";
|
|
3851 }
|
|
3852
|
|
3853 my $count = 0;
|
|
3854 while (1){
|
|
3855 my $header = <IN>;
|
|
3856 my $sequence= <IN>;
|
|
3857 last unless ($header and $sequence);
|
|
3858
|
|
3859 $header = fix_IDs($header); # this is to avoid problems with truncated read ID when they contain white spaces
|
|
3860
|
|
3861 ++$count;
|
|
3862
|
|
3863 if ($skip){
|
|
3864 next unless ($count > $skip);
|
|
3865 }
|
|
3866 if ($upto){
|
|
3867 last if ($count > $upto);
|
|
3868 }
|
|
3869
|
|
3870 $sequence = uc$sequence; # make input file case insensitive
|
|
3871
|
|
3872 # detecting if the input file contains tab stops, as this is likely to result in no alignments
|
|
3873 if (index($header,"\t") != -1){
|
|
3874 $seqID_contains_tabs++;
|
|
3875 }
|
|
3876
|
|
3877 ### small check if the sequence seems to be in FastA format
|
|
3878 die "Input file doesn't seem to be in FastA format at sequence $count: $!\n" unless ($header =~ /^>.*/);
|
|
3879
|
|
3880 my $sequence_C_to_T = $sequence;
|
|
3881 $sequence_C_to_T =~ tr/C/T/;
|
|
3882 print CTOT "$header$sequence_C_to_T";
|
|
3883
|
|
3884 unless ($directional){
|
|
3885 my $sequence_G_to_A = $sequence;
|
|
3886 $sequence_G_to_A =~ tr/G/A/;
|
|
3887 print GTOA "$header$sequence_G_to_A";
|
|
3888 }
|
|
3889 }
|
|
3890 if ($directional){
|
|
3891 print "\nCreated C -> T converted versions of the FastA file $filename ($count sequences in total)\n\n";
|
|
3892 }
|
|
3893 else{
|
|
3894 print "\nCreated C -> T as well as G -> A converted versions of the FastA file $filename ($count sequences in total)\n\n";
|
|
3895 }
|
|
3896 return ($C_to_T_infile,$G_to_A_infile);
|
|
3897 }
|
|
3898
|
|
3899 sub biTransformFastAFiles_paired_end {
|
|
3900 my ($file,$read_number) = @_;
|
|
3901
|
|
3902 my ($dir,$filename);
|
|
3903 if ($file =~ /\//){
|
|
3904 ($dir,$filename) = $file =~ m/(.*\/)(.*)$/;
|
|
3905 }
|
|
3906 else{
|
|
3907 $filename = $file;
|
|
3908 }
|
|
3909
|
|
3910 ### gzipped version of the infile
|
|
3911 if ($file =~ /\.gz$/){
|
|
3912 open (IN,"zcat $file |") or die "Couldn't read from file $file: $!\n";
|
|
3913 }
|
|
3914 else{
|
|
3915 open (IN,$file) or die "Couldn't read from file $file: $!\n";
|
|
3916 }
|
|
3917
|
|
3918 if ($skip){
|
|
3919 warn "Skipping the first $skip reads from $file\n";
|
|
3920 sleep (1);
|
|
3921 }
|
|
3922 if ($upto){
|
|
3923 warn "Processing reads up to sequence no. $upto from $file\n";
|
|
3924 sleep (1);
|
|
3925 }
|
|
3926
|
|
3927 my $C_to_T_infile = my $G_to_A_infile = $filename;
|
|
3928 $C_to_T_infile =~ s/$/_C_to_T.fa/;
|
|
3929 $G_to_A_infile =~ s/$/_G_to_A.fa/;
|
|
3930
|
|
3931 if ($directional){
|
|
3932 if ($read_number == 1){
|
|
3933 print "Writing a C -> T converted version of the input file $filename to $temp_dir$C_to_T_infile\n";
|
|
3934 open (CTOT,'>',"$temp_dir$C_to_T_infile") or die "Couldn't write to file $!\n";
|
|
3935 }
|
|
3936 elsif ($read_number == 2){
|
|
3937 print "Writing a G -> A converted version of the input file $filename to $temp_dir$G_to_A_infile\n";
|
|
3938 open (GTOA,'>',"$temp_dir$G_to_A_infile") or die "Couldn't write to file $!\n";
|
|
3939 }
|
|
3940 else{
|
|
3941 die "Read number needs to be 1 or 2, but was: $read_number\n\n";
|
|
3942 }
|
|
3943 }
|
|
3944 else{ # all four strand output
|
|
3945 print "Writing a C -> T converted version of the input file $filename to $temp_dir$C_to_T_infile\n";
|
|
3946 print "Writing a G -> A converted version of the input file $filename to $temp_dir$G_to_A_infile\n";
|
|
3947 open (CTOT,'>',"$temp_dir$C_to_T_infile") or die "Couldn't write to file $!\n";
|
|
3948 open (GTOA,'>',"$temp_dir$G_to_A_infile") or die "Couldn't write to file $!\n";
|
|
3949 }
|
|
3950
|
|
3951 my $count = 0;
|
|
3952
|
|
3953 while (1){
|
|
3954 my $header = <IN>;
|
|
3955 my $sequence= <IN>;
|
|
3956 last unless ($header and $sequence);
|
|
3957
|
|
3958 $header = fix_IDs($header); # this is to avoid problems with truncated read ID when they contain white spaces
|
|
3959
|
|
3960 ++$count;
|
|
3961
|
|
3962 if ($skip){
|
|
3963 next unless ($count > $skip);
|
|
3964 }
|
|
3965 if ($upto){
|
|
3966 last if ($count > $upto);
|
|
3967 }
|
|
3968
|
|
3969 $sequence = uc$sequence; # make input file case insensitive
|
|
3970
|
|
3971 # detecting if the input file contains tab stops, as this is likely to result in no alignments
|
|
3972 if (index($header,"\t") != -1){
|
|
3973 $seqID_contains_tabs++;
|
|
3974 }
|
|
3975
|
|
3976 ## small check if the sequence seems to be in FastA format
|
|
3977 die "Input file doesn't seem to be in FastA format at sequence $count: $!\n" unless ($header =~ /^>.*/);
|
|
3978
|
|
3979 if ($read_number == 1){
|
|
3980 if ($bowtie2){
|
|
3981 $header =~ s/$/\/1\/1/;
|
|
3982 }
|
|
3983 else{
|
|
3984 $header =~ s/$/\/1/;
|
|
3985 }
|
|
3986 }
|
|
3987 elsif ($read_number == 2){
|
|
3988 if ($bowtie2){
|
|
3989 $header =~ s/$/\/2\/2/;
|
|
3990 }
|
|
3991 else{
|
|
3992 $header =~ s/$/\/2/;
|
|
3993 }
|
|
3994 }
|
|
3995 else{
|
|
3996 die "Read number needs to be 1 or 2, but was: $read_number\n\n";
|
|
3997 }
|
|
3998 my $sequence_C_to_T = my $sequence_G_to_A = $sequence;
|
|
3999
|
|
4000 $sequence_C_to_T =~ tr/C/T/;
|
|
4001 $sequence_G_to_A =~ tr/G/A/;
|
|
4002
|
|
4003 if ($directional){
|
|
4004
|
|
4005 if ($read_number == 1){
|
|
4006 print CTOT "$header$sequence_C_to_T";
|
|
4007 }
|
|
4008 elsif ($read_number == 2){
|
|
4009 print GTOA "$header$sequence_G_to_A";
|
|
4010 }
|
|
4011 }
|
|
4012 else{
|
|
4013 print CTOT "$header$sequence_C_to_T";
|
|
4014 print GTOA "$header$sequence_G_to_A";
|
|
4015 }
|
|
4016 }
|
|
4017
|
|
4018 if ($directional){
|
|
4019 if ($read_number == 1){
|
|
4020 print "\nCreated C -> T converted version of the FastA file $filename ($count sequences in total)\n\n";
|
|
4021 }
|
|
4022 else{
|
|
4023 print "\nCreated G -> A converted version of the FastA file $filename ($count sequences in total)\n\n";
|
|
4024 }
|
|
4025 }
|
|
4026 else{
|
|
4027 print "\nCreated C -> T as well as G -> A converted versions of the FastA file $filename ($count sequences in total)\n\n";
|
|
4028 }
|
|
4029
|
|
4030 if ($directional){
|
|
4031 if ($read_number == 1){
|
|
4032 return ($C_to_T_infile);
|
|
4033 }
|
|
4034 else{
|
|
4035 return ($G_to_A_infile);
|
|
4036 }
|
|
4037 }
|
|
4038 else{
|
|
4039 return ($C_to_T_infile,$G_to_A_infile);
|
|
4040 }
|
|
4041 }
|
|
4042
|
|
4043
|
|
4044 sub biTransformFastQFiles {
|
|
4045 my $file = shift;
|
|
4046 my ($dir,$filename);
|
|
4047 if ($file =~ /\//){
|
|
4048 ($dir,$filename) = $file =~ m/(.*\/)(.*)$/;
|
|
4049 }
|
|
4050 else{
|
|
4051 $filename = $file;
|
|
4052 }
|
|
4053
|
|
4054 ### gzipped version of the infile
|
|
4055 if ($file =~ /\.gz$/){
|
|
4056 open (IN,"zcat $file |") or die "Couldn't read from file $file: $!\n";
|
|
4057 }
|
|
4058 else{
|
|
4059 open (IN,$file) or die "Couldn't read from file $file: $!\n";
|
|
4060 }
|
|
4061
|
|
4062 if ($skip){
|
|
4063 warn "Skipping the first $skip reads from $file\n";
|
|
4064 sleep (1);
|
|
4065 }
|
|
4066 if ($upto){
|
|
4067 warn "Processing reads up to sequence no. $upto from $file\n";
|
|
4068 sleep (1);
|
|
4069 }
|
|
4070
|
|
4071 my $C_to_T_infile = my $G_to_A_infile = $filename;
|
|
4072
|
|
4073 $C_to_T_infile =~ s/$/_C_to_T.fastq/;
|
|
4074 print "Writing a C -> T converted version of the input file $filename to $temp_dir$C_to_T_infile\n";
|
|
4075 open (CTOT,'>',"$temp_dir$C_to_T_infile") or die "Couldn't write to file $!\n";
|
|
4076
|
|
4077 unless ($directional){
|
|
4078 $G_to_A_infile =~ s/$/_G_to_A.fastq/;
|
|
4079 print "Writing a G -> A converted version of the input file $filename to $temp_dir$G_to_A_infile\n";
|
|
4080 open (GTOA,'>',"$temp_dir$G_to_A_infile") or die "Couldn't write to file $!\n";
|
|
4081 }
|
|
4082
|
|
4083 my $count = 0;
|
|
4084 while (1){
|
|
4085 my $identifier = <IN>;
|
|
4086 my $sequence = <IN>;
|
|
4087 my $identifier2 = <IN>;
|
|
4088 my $quality_score = <IN>;
|
|
4089 last unless ($identifier and $sequence and $identifier2 and $quality_score);
|
|
4090
|
|
4091 $identifier = fix_IDs($identifier); # this is to avoid problems with truncated read ID when they contain white spaces
|
|
4092
|
|
4093 ++$count;
|
|
4094
|
|
4095 if ($skip){
|
|
4096 next unless ($count > $skip);
|
|
4097 }
|
|
4098 if ($upto){
|
|
4099 last if ($count > $upto);
|
|
4100 }
|
|
4101
|
|
4102 $sequence = uc$sequence; # make input file case insensitive
|
|
4103
|
|
4104 # detecting if the input file contains tab stops, as this is likely to result in no alignments
|
|
4105 if (index($identifier,"\t") != -1){
|
|
4106 $seqID_contains_tabs++;
|
|
4107 }
|
|
4108
|
|
4109 ## small check if the sequence file appears to be a FastQ file
|
|
4110 if ($identifier !~ /^\@/ or $identifier2 !~ /^\+/){
|
|
4111 die "Input file doesn't seem to be in FastQ format at sequence $count: $!\n";
|
|
4112 }
|
|
4113
|
|
4114 my $sequence_C_to_T = $sequence;
|
|
4115 $sequence_C_to_T =~ tr/C/T/;
|
|
4116 print CTOT join ('',$identifier,$sequence_C_to_T,$identifier2,$quality_score);
|
|
4117
|
|
4118 unless ($directional){
|
|
4119 my $sequence_G_to_A = $sequence;
|
|
4120 $sequence_G_to_A =~ tr/G/A/;
|
|
4121 print GTOA join ('',$identifier,$sequence_G_to_A,$identifier2,$quality_score);
|
|
4122 }
|
|
4123 }
|
|
4124
|
|
4125 if ($directional){
|
|
4126 print "\nCreated C -> T converted versions of the FastQ file $filename ($count sequences in total)\n\n";
|
|
4127 }
|
|
4128 else{
|
|
4129 print "\nCreated C -> T as well as G -> A converted versions of the FastQ file $filename ($count sequences in total)\n\n";
|
|
4130 }
|
|
4131
|
|
4132 return ($C_to_T_infile,$G_to_A_infile);
|
|
4133 }
|
|
4134
|
|
4135 sub biTransformFastQFiles_paired_end {
|
|
4136 my ($file,$read_number) = @_;
|
|
4137 my ($dir,$filename);
|
|
4138
|
|
4139 if ($file =~ /\//){
|
|
4140 ($dir,$filename) = $file =~ m/(.*\/)(.*)$/;
|
|
4141 }
|
|
4142 else{
|
|
4143 $filename = $file;
|
|
4144 }
|
|
4145
|
|
4146 ### gzipped version of the infile
|
|
4147 if ($file =~ /\.gz$/){
|
|
4148 open (IN,"zcat $file |") or die "Couldn't read from file $file: $!\n";
|
|
4149 }
|
|
4150 else{
|
|
4151 open (IN,$file) or die "Couldn't read from file $file: $!\n";
|
|
4152 }
|
|
4153
|
|
4154 if ($skip){
|
|
4155 warn "Skipping the first $skip reads from $file\n";
|
|
4156 sleep (1);
|
|
4157 }
|
|
4158 if ($upto){
|
|
4159 warn "Processing reads up to sequence no. $upto from $file\n";
|
|
4160 sleep (1);
|
|
4161 }
|
|
4162
|
|
4163 my $C_to_T_infile = my $G_to_A_infile = $filename;
|
|
4164 $C_to_T_infile =~ s/$/_C_to_T.fastq/;
|
|
4165 $G_to_A_infile =~ s/$/_G_to_A.fastq/;
|
|
4166
|
|
4167 if ($directional){
|
|
4168 if ($read_number == 1){
|
|
4169 print "Writing a C -> T converted version of the input file $filename to $temp_dir$C_to_T_infile\n";
|
|
4170 open (CTOT,'>',"$temp_dir$C_to_T_infile") or die "Couldn't write to file $!\n";
|
|
4171 }
|
|
4172 elsif ($read_number == 2){
|
|
4173 print "Writing a G -> A converted version of the input file $filename to $temp_dir$G_to_A_infile\n";
|
|
4174 open (GTOA,'>',"$temp_dir$G_to_A_infile") or die "Couldn't write to file $!\n";
|
|
4175 }
|
|
4176 else{
|
|
4177 die "Read number needs to be 1 or 2, but was $read_number!\n\n";
|
|
4178 }
|
|
4179 }
|
|
4180 else{
|
|
4181 print "Writing a C -> T converted version of the input file $filename to $temp_dir$C_to_T_infile\n";
|
|
4182 print "Writing a G -> A converted version of the input file $filename to $temp_dir$G_to_A_infile\n";
|
|
4183 open (CTOT,'>',"$temp_dir$C_to_T_infile") or die "Couldn't write to file $!\n";
|
|
4184 open (GTOA,'>',"$temp_dir$G_to_A_infile") or die "Couldn't write to file $!\n";
|
|
4185 }
|
|
4186
|
|
4187 my $count = 0;
|
|
4188
|
|
4189 while (1){
|
|
4190 my $identifier = <IN>;
|
|
4191 my $sequence = <IN>;
|
|
4192 my $identifier2 = <IN>;
|
|
4193 my $quality_score = <IN>;
|
|
4194 last unless ($identifier and $sequence and $identifier2 and $quality_score);
|
|
4195 ++$count;
|
|
4196
|
|
4197 $identifier = fix_IDs($identifier); # this is to avoid problems with truncated read ID when they contain white spaces
|
|
4198
|
|
4199 if ($skip){
|
|
4200 next unless ($count > $skip);
|
|
4201 }
|
|
4202 if ($upto){
|
|
4203 last if ($count > $upto);
|
|
4204 }
|
|
4205
|
|
4206 $sequence= uc$sequence; # make input file case insensitive
|
|
4207
|
|
4208 ## small check if the sequence file appears to be a FastQ file
|
|
4209 if ($identifier !~ /^\@/ or $identifier2 !~ /^\+/){
|
|
4210 die "Input file doesn't seem to be in FastQ format at sequence $count: $!\n";
|
|
4211 }
|
|
4212 my $sequence_C_to_T = my $sequence_G_to_A = $sequence;
|
|
4213
|
|
4214 if ($read_number == 1){
|
|
4215 if ($bowtie2){
|
|
4216 $identifier =~ s/$/\/1\/1/;
|
|
4217 }
|
|
4218 else{
|
|
4219 $identifier =~ s/$/\/1/;
|
|
4220 }
|
|
4221 }
|
|
4222 elsif ($read_number == 2){
|
|
4223 if ($bowtie2){
|
|
4224 $identifier =~ s/$/\/2\/2/;
|
|
4225 }
|
|
4226 else{
|
|
4227 $identifier =~ s/$/\/2/;
|
|
4228 }
|
|
4229 }
|
|
4230 else{
|
|
4231 die "Read number needs to be 1 or 2\n";
|
|
4232 }
|
|
4233
|
|
4234 $sequence_C_to_T =~ tr/C/T/;
|
|
4235 $sequence_G_to_A =~ tr/G/A/;
|
|
4236
|
|
4237 if ($directional){
|
|
4238 if ($read_number == 1){
|
|
4239 print CTOT join ('',$identifier,$sequence_C_to_T,$identifier2,$quality_score);
|
|
4240 }
|
|
4241 else{
|
|
4242 print GTOA join ('',$identifier,$sequence_G_to_A,$identifier2,$quality_score);
|
|
4243 }
|
|
4244 }
|
|
4245 else{
|
|
4246 print CTOT join ('',$identifier,$sequence_C_to_T,$identifier2,$quality_score);
|
|
4247 print GTOA join ('',$identifier,$sequence_G_to_A,$identifier2,$quality_score);
|
|
4248 }
|
|
4249 }
|
|
4250
|
|
4251 if ($directional){
|
|
4252 if ($read_number == 1){
|
|
4253 print "\nCreated C -> T converted version of the FastQ file $filename ($count sequences in total)\n\n";
|
|
4254 }
|
|
4255 else{
|
|
4256 print "\nCreated G -> A converted version of the FastQ file $filename ($count sequences in total)\n\n";
|
|
4257 }
|
|
4258 }
|
|
4259 else{
|
|
4260 print "\nCreated C -> T as well as G -> A converted versions of the FastQ file $filename ($count sequences in total)\n\n";
|
|
4261 }
|
|
4262 if ($directional){
|
|
4263 if ($read_number == 1){
|
|
4264 return ($C_to_T_infile);
|
|
4265 }
|
|
4266 else{
|
|
4267 return ($G_to_A_infile);
|
|
4268 }
|
|
4269 }
|
|
4270 else{
|
|
4271 return ($C_to_T_infile,$G_to_A_infile);
|
|
4272 }
|
|
4273 }
|
|
4274
|
|
4275 sub fix_IDs{
|
|
4276 my $id = shift;
|
|
4277 $id =~ s/[ \t]+/_/g; # replace spaces or tabs with underscores
|
|
4278 return $id;
|
|
4279 }
|
|
4280
|
|
4281 sub ensure_sensical_alignment_orientation_single_end{
|
|
4282 my $index = shift; # index number if the sequence produced an alignment
|
|
4283 my $strand = shift;
|
|
4284 ### setting $orientation to 1 if it is in the correct orientation, and leave it 0 if it is the nonsensical wrong one
|
|
4285 my $orientation = 0;
|
|
4286 ##############################################################################################################
|
|
4287 ## FORWARD converted read against FORWARD converted genome (read: C->T.....C->T.. genome:C->T.......C->T)
|
|
4288 ## here we only want reads in the forward (+) orientation
|
|
4289 if ($fhs[$index]->{name} eq 'CTreadCTgenome') {
|
|
4290 ### if the alignment is (+) we count it, and return 1 for a correct orientation
|
|
4291 if ($strand eq '+') {
|
|
4292 $fhs[$index]->{seen}++;
|
|
4293 $orientation = 1;
|
|
4294 return $orientation;
|
|
4295 }
|
|
4296 ### if the orientation equals (-) the alignment is nonsensical
|
|
4297 elsif ($strand eq '-') {
|
|
4298 $fhs[$index]->{wrong_strand}++;
|
|
4299 return $orientation;
|
|
4300 }
|
|
4301 }
|
|
4302 ###############################################################################################################
|
|
4303 ## FORWARD converted read against reverse converted genome (read: C->T.....C->T.. genome: G->A.......G->A)
|
|
4304 ## here we only want reads in the forward (-) orientation
|
|
4305 elsif ($fhs[$index]->{name} eq 'CTreadGAgenome') {
|
|
4306 ### if the alignment is (-) we count it and return 1 for a correct orientation
|
|
4307 if ($strand eq '-') {
|
|
4308 $fhs[$index]->{seen}++;
|
|
4309 $orientation = 1;
|
|
4310 return $orientation;
|
|
4311 }
|
|
4312 ### if the orientation equals (+) the alignment is nonsensical
|
|
4313 elsif ($strand eq '+') {
|
|
4314 $fhs[$index]->{wrong_strand}++;
|
|
4315 return $orientation;
|
|
4316 }
|
|
4317 }
|
|
4318 ###############################################################################################################
|
|
4319 ## Reverse converted read against FORWARD converted genome (read: G->A.....G->A.. genome: C->T.......C->T)
|
|
4320 ## here we only want reads in the forward (-) orientation
|
|
4321 elsif ($fhs[$index]->{name} eq 'GAreadCTgenome') {
|
|
4322 ### if the alignment is (-) we count it and return 1 for a correct orientation
|
|
4323 if ($strand eq '-') {
|
|
4324 $fhs[$index]->{seen}++;
|
|
4325 $orientation = 1;
|
|
4326 return $orientation;
|
|
4327 }
|
|
4328 ### if the orientation equals (+) the alignment is nonsensical
|
|
4329 elsif ($strand eq '+') {
|
|
4330 $fhs[$index]->{wrong_strand}++;
|
|
4331 return $orientation;
|
|
4332 }
|
|
4333 }
|
|
4334 ###############################################################################################################
|
|
4335 ## Reverse converted read against reverse converted genome (read: G->A.....G->A.. genome: G->A.......G->A)
|
|
4336 ## here we only want reads in the forward (+) orientation
|
|
4337 elsif ($fhs[$index]->{name} eq 'GAreadGAgenome') {
|
|
4338 ### if the alignment is (+) we count it and return 1 for a correct orientation
|
|
4339 if ($strand eq '+') {
|
|
4340 $fhs[$index]->{seen}++;
|
|
4341 $orientation = 1;
|
|
4342 return $orientation;
|
|
4343 }
|
|
4344 ### if the orientation equals (-) the alignment is nonsensical
|
|
4345 elsif ($strand eq '-') {
|
|
4346 $fhs[$index]->{wrong_strand}++;
|
|
4347 return $orientation;
|
|
4348 }
|
|
4349 } else{
|
|
4350 die "One of the above conditions must be true\n";
|
|
4351 }
|
|
4352 }
|
|
4353
|
|
4354 sub ensure_sensical_alignment_orientation_paired_ends{
|
|
4355 my ($index,$id_1,$strand_1,$id_2,$strand_2) = @_; # index number if the sequence produced an alignment
|
|
4356 ### setting $orientation to 1 if it is in the correct orientation, and leave it 0 if it is the nonsensical wrong one
|
|
4357 my $orientation = 0;
|
|
4358 ##############################################################################################################
|
|
4359 ## [Index 0, sequence originated from (converted) forward strand]
|
|
4360 ## CT converted read 1
|
|
4361 ## GA converted read 2
|
|
4362 ## CT converted genome
|
|
4363 ## here we only want read 1 in (+) orientation and read 2 in (-) orientation
|
|
4364 if ($fhs[$index]->{name} eq 'CTread1GAread2CTgenome') {
|
|
4365 ### if the paired-end alignment is read1 (+) and read2 (-) we count it, and return 1 for a correct orientation
|
|
4366 if ($id_1 =~ /1$/ and $strand_1 eq '+' and $id_2 =~ /2$/ and $strand_2 eq '-') {
|
|
4367 $fhs[$index]->{seen}++;
|
|
4368 $orientation = 1;
|
|
4369 return $orientation;
|
|
4370 }
|
|
4371 ### if the read 2 is in (+) orientation and read 1 in (-) the alignment is nonsensical
|
|
4372 elsif ($id_1 =~ /2$/ and $strand_1 eq '+' and $id_2 =~ /1$/ and $strand_2 eq '-') {
|
|
4373 $fhs[$index]->{wrong_strand}++;
|
|
4374 return $orientation;
|
|
4375 }
|
|
4376 else{
|
|
4377 die "id1: $id_1\tid2: $id_2\tThis should be impossible\n";
|
|
4378 }
|
|
4379 }
|
|
4380 ###############################################################################################################
|
|
4381 ## [Index 1, sequence originated from (converted) reverse strand]
|
|
4382 ## GA converted read 1
|
|
4383 ## CT converted read 2
|
|
4384 ## GA converted genome
|
|
4385 ## here we only want read 1 in (+) orientation and read 2 in (-) orientation
|
|
4386 elsif ($fhs[$index]->{name} eq 'GAread1CTread2GAgenome') {
|
|
4387 ### if the paired-end alignment is read1 (+) and read2 (-) we count it, and return 1 for a correct orientation
|
|
4388 if ($id_1 =~ /1$/ and $strand_1 eq '+' and $id_2 =~ /2$/ and $strand_2 eq '-') {
|
|
4389 $fhs[$index]->{seen}++;
|
|
4390 $orientation = 1;
|
|
4391 return $orientation;
|
|
4392 }
|
|
4393 ### if the read 2 is in (+) orientation and read 1 in (-) the alignment is nonsensical
|
|
4394 elsif ($id_1 =~ /2$/ and $strand_1 eq '+' and $id_2 =~ /1$/ and $strand_2 eq '-') {
|
|
4395 $fhs[$index]->{wrong_strand}++;
|
|
4396 return $orientation;
|
|
4397 }
|
|
4398 else{
|
|
4399 die "id1: $id_1\tid2: $id_2\tThis should be impossible\n";
|
|
4400 }
|
|
4401 }
|
|
4402 ###############################################################################################################
|
|
4403 ## [Index 2, sequence originated from complementary to (converted) forward strand]
|
|
4404 ## GA converted read 1
|
|
4405 ## CT converted read 2
|
|
4406 ## CT converted genome
|
|
4407 ## here we only want read 1 in (-) orientation and read 2 in (+) orientation
|
|
4408 elsif ($fhs[$index]->{name} eq 'GAread1CTread2CTgenome') {
|
|
4409 ### if the paired-end alignment is read1 (-) and read2 (+) we count it, and return 1 for a correct orientation
|
|
4410 if ($id_1 =~ /2$/ and $strand_1 eq '+' and $id_2 =~ /1$/ and $strand_2 eq '-') {
|
|
4411 $fhs[$index]->{seen}++;
|
|
4412 $orientation = 1;
|
|
4413 return $orientation;
|
|
4414 }
|
|
4415 ### if the read 2 is in (+) orientation and read 1 in (-) the alignment is nonsensical
|
|
4416 elsif ($id_1 =~ /1$/ and $strand_1 eq '+' and $id_2 =~ /2$/ and $strand_2 eq '-') {
|
|
4417 $fhs[$index]->{wrong_strand}++;
|
|
4418 return $orientation;
|
|
4419 }
|
|
4420 else{
|
|
4421 die "id1: $id_1\tid2: $id_2\tThis should be impossible\n";
|
|
4422 }
|
|
4423 }
|
|
4424 ###############################################################################################################
|
|
4425 ## [Index 3, sequence originated from complementary to (converted) reverse strand]
|
|
4426 ## CT converted read 1
|
|
4427 ## GA converted read 2
|
|
4428 ## GA converted genome
|
|
4429 ## here we only want read 1 in (+) orientation and read 2 in (-) orientation
|
|
4430 elsif ($fhs[$index]->{name} eq 'CTread1GAread2GAgenome') {
|
|
4431 ### if the paired-end alignment is read1 (-) and read2 (+) we count it, and return 1 for a correct orientation
|
|
4432 if ($id_1 =~ /2$/ and $strand_1 eq '+' and $id_2 =~ /1$/ and $strand_2 eq '-') {
|
|
4433 $fhs[$index]->{seen}++;
|
|
4434 $orientation = 1;
|
|
4435 return $orientation;
|
|
4436 }
|
|
4437 ### if the read 2 is in (+) orientation and read 1 in (-) the alignment is nonsensical
|
|
4438 elsif ($id_1 =~ /1$/ and $strand_1 eq '+' and $id_2 =~ /2$/ and $strand_2 eq '-') {
|
|
4439 $fhs[$index]->{wrong_strand}++;
|
|
4440 return $orientation;
|
|
4441 }
|
|
4442 else{
|
|
4443 die "id1: $id_1\tid2: $id_2\tThis should be impossible\n";
|
|
4444 }
|
|
4445 }
|
|
4446 else{
|
|
4447 die "One of the above conditions must be true\n";
|
|
4448 }
|
|
4449 }
|
|
4450
|
|
4451 #####################################################################################################################################################
|
|
4452
|
|
4453 ### Bowtie 1 (default) | PAIRED-END | FASTA
|
|
4454
|
|
4455 sub paired_end_align_fragments_to_bisulfite_genome_fastA {
|
|
4456
|
|
4457 my ($C_to_T_infile_1,$G_to_A_infile_1,$C_to_T_infile_2,$G_to_A_infile_2) = @_;
|
|
4458
|
|
4459 if ($directional){
|
|
4460 print "Input files are $C_to_T_infile_1 and $G_to_A_infile_2 (FastA)\n";
|
|
4461 }
|
|
4462 else{
|
|
4463 print "Input files are $C_to_T_infile_1 and $G_to_A_infile_1 and $C_to_T_infile_2 and $G_to_A_infile_2 (FastA)\n";
|
|
4464 }
|
|
4465
|
|
4466 ## Now starting up to 4 instances of Bowtie feeding in the converted sequence files and reading in the first line of the bowtie output, and storing it in the
|
|
4467 ## data structure above
|
|
4468 if ($directional){
|
|
4469 warn "Now running 2 instances of Bowtie against the bisulfite genome of $genome_folder with the specified options: $bowtie_options\n\n";
|
|
4470 }
|
|
4471 else{
|
|
4472 warn "Now running 4 individual instances of Bowtie against the bisulfite genome of $genome_folder with the specified options: $bowtie_options\n\n";
|
|
4473 }
|
|
4474
|
|
4475 foreach my $fh (@fhs) {
|
|
4476
|
|
4477 if ($directional){
|
|
4478 unless ($fh->{inputfile_1}){
|
|
4479 $fh->{last_seq_id} = undef;
|
|
4480 $fh->{last_line_1} = undef;
|
|
4481 $fh->{last_line_2} = undef;
|
|
4482 next;
|
|
4483 }
|
|
4484 }
|
|
4485
|
|
4486 my $bt_options = $bowtie_options;
|
|
4487 if ($fh->{name} eq 'CTread1GAread2CTgenome' or $fh->{name} eq 'GAread1CTread2GAgenome'){
|
|
4488 $bt_options .= ' --norc'; ### ensuring the alignments are only reported in a sensible manner
|
|
4489 }
|
|
4490 else {
|
|
4491 $bt_options .= ' --nofw';
|
|
4492 }
|
|
4493
|
|
4494 warn "Now starting a Bowtie paired-end alignment for $fh->{name} (reading in sequences from $temp_dir$fh->{inputfile_1} and $temp_dir$fh->{inputfile_2}, with the options: $bt_options)\n";
|
|
4495 open ($fh->{fh},"$path_to_bowtie $bt_options $fh->{bisulfiteIndex} -1 $temp_dir$fh->{inputfile_1} -2 $temp_dir$fh->{inputfile_2} |") or die "Can't open pipe to bowtie: $!";
|
|
4496
|
|
4497 my $line_1 = $fh->{fh}->getline();
|
|
4498 my $line_2 = $fh->{fh}->getline();
|
|
4499
|
|
4500 # if Bowtie produces an alignment we store the first line of the output
|
|
4501 if ($line_1 and $line_2) {
|
|
4502 chomp $line_1;
|
|
4503 chomp $line_2;
|
|
4504 my $id_1 = (split(/\t/,$line_1))[0]; # this is the first element of the first bowtie output line (= the sequence identifier)
|
|
4505 my $id_2 = (split(/\t/,$line_2))[0]; # this is the first element of the second bowtie output line
|
|
4506
|
|
4507 ### Bowtie always reports the alignment with the smaller chromosomal position first. This can be either sequence 1 or sequence 2.
|
|
4508 ### We will thus identify which sequence was read 1 and store this ID as last_seq_id
|
|
4509
|
|
4510 if ($id_1 =~ s/\/1$//){ # removing the read 1 tag if present
|
|
4511 $fh->{last_seq_id} = $id_1;
|
|
4512 }
|
|
4513 elsif ($id_2 =~ s/\/1$//){ # removing the read 1 tag if present
|
|
4514 $fh->{last_seq_id} = $id_2;
|
|
4515 }
|
|
4516 else{
|
|
4517 die "Either the first or the second id need to be read 1! ID1 was: $id_1; ID2 was: $id_2\n";
|
|
4518 }
|
|
4519
|
|
4520 $fh->{last_line_1} = $line_1; # this contains either read 1 or read 2
|
|
4521 $fh->{last_line_2} = $line_2; # this contains either read 1 or read 2
|
|
4522 warn "Found first alignment:\n$fh->{last_line_1}\n$fh->{last_line_2}\n";
|
|
4523 }
|
|
4524 # otherwise we just initialise last_seq_id and last_lines as undefined
|
|
4525 else {
|
|
4526 print "Found no alignment, assigning undef to last_seq_id and last_lines\n";
|
|
4527 $fh->{last_seq_id} = undef;
|
|
4528 $fh->{last_line_1} = undef;
|
|
4529 $fh->{last_line_2} = undef;
|
|
4530 }
|
|
4531 }
|
|
4532 }
|
|
4533
|
|
4534 ### Bowtie 2 | PAIRED-END | FASTA
|
|
4535
|
|
4536 sub paired_end_align_fragments_to_bisulfite_genome_fastA_bowtie2 {
|
|
4537 my ($C_to_T_infile_1,$G_to_A_infile_1,$C_to_T_infile_2,$G_to_A_infile_2) = @_;
|
|
4538 if ($directional){
|
|
4539 print "Input files are $C_to_T_infile_1 and $G_to_A_infile_2 (FastA)\n";
|
|
4540 }
|
|
4541 else{
|
|
4542 print "Input files are $C_to_T_infile_1 and $G_to_A_infile_1 and $C_to_T_infile_2 and $G_to_A_infile_2 (FastA)\n";
|
|
4543 }
|
|
4544
|
|
4545 ## Now starting up to 4 instances of Bowtie feeding in the converted sequence files and reading in the first line of the bowtie output, and storing it in the
|
|
4546 ## data structure above
|
|
4547 if ($directional){
|
|
4548 warn "Now running 2 instances of Bowtie 2 against the bisulfite genome of $genome_folder with the specified options: $bowtie_options\n\n";
|
|
4549 }
|
|
4550 else{
|
|
4551 warn "Now running 4 individual instances of Bowtie 2 against the bisulfite genome of $genome_folder with the specified options: $bowtie_options\n\n";
|
|
4552 }
|
|
4553
|
|
4554 foreach my $fh (@fhs) {
|
|
4555
|
|
4556 if ($directional){
|
|
4557 unless ($fh->{inputfile_1}){
|
|
4558 $fh->{last_seq_id} = undef;
|
|
4559 $fh->{last_line_1} = undef;
|
|
4560 $fh->{last_line_2} = undef;
|
|
4561 next;
|
|
4562 }
|
|
4563 }
|
|
4564
|
|
4565 my $bt2_options = $bowtie_options;
|
|
4566 if ($fh->{name} eq 'CTread1GAread2CTgenome' or $fh->{name} eq 'GAread1CTread2GAgenome'){
|
|
4567 $bt2_options .= ' --norc'; ### ensuring the alignments are only reported in a sensible manner
|
|
4568 }
|
|
4569 else {
|
|
4570 $bt2_options .= ' --nofw';
|
|
4571 }
|
|
4572
|
|
4573 warn "Now starting a Bowtie 2 paired-end alignment for $fh->{name} (reading in sequences from $temp_dir$fh->{inputfile_1} and $temp_dir$fh->{inputfile_2}, with the options: $bt2_options))\n";
|
|
4574 open ($fh->{fh},"$path_to_bowtie $bt2_options $fh->{bisulfiteIndex} -1 $temp_dir$fh->{inputfile_1} -2 $temp_dir$fh->{inputfile_2} |") or die "Can't open pipe to bowtie: $!";
|
|
4575
|
|
4576 ### Bowtie 2 outputs out SAM format, so we need to skip everything until the first sequence
|
|
4577 while (1){
|
|
4578 $_ = $fh->{fh}->getline();
|
|
4579 if ($_) {
|
|
4580 last unless ($_ =~ /^\@/); # SAM headers start with @
|
|
4581 }
|
|
4582 else{
|
|
4583 last; # no alignment output
|
|
4584 }
|
|
4585 }
|
|
4586
|
|
4587 my $line_1 = $_;
|
|
4588 my $line_2 = $fh->{fh}->getline();
|
|
4589
|
|
4590 # if Bowtie produces an alignment we store the first line of the output
|
|
4591 if ($line_1 and $line_2) {
|
|
4592 chomp $line_1;
|
|
4593 chomp $line_2;
|
|
4594 my $id_1 = (split(/\t/,$line_1))[0]; # this is the first element of the first bowtie output line (= the sequence identifier)
|
|
4595 my $id_2 = (split(/\t/,$line_2))[0]; # this is the first element of the second bowtie output line
|
|
4596
|
|
4597 ### Bowtie always reports the alignment with the smaller chromosomal position first. This can be either sequence 1 or sequence 2.
|
|
4598 ### We will thus identify which sequence was read 1 and store this ID as last_seq_id
|
|
4599
|
|
4600 if ($id_1 =~ s/\/1$//){ # removing the read 1 /1 tag if present (remember that Bowtie2 clips off /1 or /2 line endings itself, so we added /1/1 or /2/2 to start with
|
|
4601 $fh->{last_seq_id} = $id_1;
|
|
4602 }
|
|
4603 elsif ($id_2 =~ s/\/1$//){ # removing the read 1 /2 tag if present
|
|
4604 $fh->{last_seq_id} = $id_2;
|
|
4605 }
|
|
4606 else{
|
|
4607 warn "Either the first or the second id need to be read 1! ID1 was: $id_1; ID2 was: $id_2\n";
|
|
4608 }
|
|
4609
|
|
4610 $fh->{last_line_1} = $line_1; # this contains either read 1 or read 2
|
|
4611 $fh->{last_line_2} = $line_2; # this contains either read 1 or read 2
|
|
4612 warn "Found first alignment:\n$fh->{last_line_1}\n$fh->{last_line_2}\n";
|
|
4613 }
|
|
4614 # otherwise we just initialise last_seq_id and last_lines as undefined
|
|
4615 else {
|
|
4616 print "Found no alignment, assigning undef to last_seq_id and last_lines\n";
|
|
4617 $fh->{last_seq_id} = undef;
|
|
4618 $fh->{last_line_1} = undef;
|
|
4619 $fh->{last_line_2} = undef;
|
|
4620 }
|
|
4621 }
|
|
4622 }
|
|
4623
|
|
4624 ### Bowtie 1 (default) | PAIRED-END | FASTQ
|
|
4625
|
|
4626 sub paired_end_align_fragments_to_bisulfite_genome_fastQ {
|
|
4627 my ($C_to_T_infile_1,$G_to_A_infile_1,$C_to_T_infile_2,$G_to_A_infile_2) = @_;
|
|
4628 if ($directional){
|
|
4629 print "Input files are $C_to_T_infile_1 $G_to_A_infile_2 (FastQ)\n";
|
|
4630 }
|
|
4631 else{
|
|
4632 print "Input files are $C_to_T_infile_1 and $G_to_A_infile_1 and $C_to_T_infile_2 and $G_to_A_infile_2 (FastQ)\n";
|
|
4633 }
|
|
4634
|
|
4635 ## Now starting up to 4 instances of Bowtie feeding in the converted sequence files and reading in the first line of the bowtie output, and storing it in the
|
|
4636 ## data structure above
|
|
4637 if ($directional){
|
|
4638 warn "Now running 2 instances of Bowtie against the bisulfite genome of $genome_folder with the specified options: $bowtie_options\n\n";
|
|
4639 }
|
|
4640 else{
|
|
4641 warn "Now running 4 individual instances of Bowtie against the bisulfite genome of $genome_folder with the specified options: $bowtie_options\n\n";
|
|
4642 }
|
|
4643
|
|
4644 foreach my $fh (@fhs) {
|
|
4645
|
|
4646 if ($directional){
|
|
4647 unless ($fh->{inputfile_1}){
|
|
4648 $fh->{last_seq_id} = undef;
|
|
4649 $fh->{last_line_1} = undef;
|
|
4650 $fh->{last_line_2} = undef;
|
|
4651 next;
|
|
4652 }
|
|
4653 }
|
|
4654
|
|
4655 my $bt_options = $bowtie_options;
|
|
4656 if ($fh->{name} eq 'CTread1GAread2CTgenome' or $fh->{name} eq 'GAread1CTread2GAgenome'){
|
|
4657 $bt_options .= ' --norc'; ### ensuring the alignments are only reported in a sensible manner
|
|
4658 }
|
|
4659 else {
|
|
4660 $bt_options .= ' --nofw';
|
|
4661 }
|
|
4662
|
|
4663 warn "Now starting a Bowtie paired-end alignment for $fh->{name} (reading in sequences from $temp_dir$fh->{inputfile_1} and $temp_dir$fh->{inputfile_2}, with the options: $bt_options))\n";
|
|
4664 open ($fh->{fh},"$path_to_bowtie $bt_options $fh->{bisulfiteIndex} -1 $temp_dir$fh->{inputfile_1} -2 $temp_dir$fh->{inputfile_2} |") or die "Can't open pipe to bowtie: $!";
|
|
4665
|
|
4666 my $line_1 = $fh->{fh}->getline();
|
|
4667 my $line_2 = $fh->{fh}->getline();
|
|
4668
|
|
4669 # if Bowtie produces an alignment we store the first line of the output
|
|
4670 if ($line_1 and $line_2) {
|
|
4671 chomp $line_1;
|
|
4672 chomp $line_2;
|
|
4673 ### Bowtie always reports the alignment with the smaller chromosomal position first. This can be either sequence 1 or sequence 2.
|
|
4674 ### We will thus identify which sequence was read 1 and store this ID as last_seq_id
|
|
4675
|
|
4676 my $id_1 = (split(/\t/,$line_1))[0]; # this is the first element of the first bowtie output line (= the sequence identifier)
|
|
4677 my $id_2 = (split(/\t/,$line_2))[0]; # this is the first element of the second bowtie output line
|
|
4678
|
|
4679 if ($id_1 =~ s/\/1$//){ # removing the read 1 tag if present
|
|
4680 $fh->{last_seq_id} = $id_1;
|
|
4681 }
|
|
4682 elsif ($id_2 =~ s/\/1$//){ # removing the read 1 tag if present
|
|
4683 $fh->{last_seq_id} = $id_2;
|
|
4684 }
|
|
4685 else{
|
|
4686 die "Either the first or the second id need to be read 1! ID1 was: $id_1; ID2 was: $id_2\n";
|
|
4687 }
|
|
4688
|
|
4689 $fh->{last_line_1} = $line_1; # this contains read 1 or read 2
|
|
4690 $fh->{last_line_2} = $line_2; # this contains read 1 or read 2
|
|
4691 warn "Found first alignment:\n$fh->{last_line_1}\n$fh->{last_line_2}\n";
|
|
4692 }
|
|
4693
|
|
4694 # otherwise we just initialise last_seq_id and last_lines as undefined
|
|
4695 else {
|
|
4696 print "Found no alignment, assigning undef to last_seq_id and last_lines\n";
|
|
4697 $fh->{last_seq_id} = undef;
|
|
4698 $fh->{last_line_1} = undef;
|
|
4699 $fh->{last_line_2} = undef;
|
|
4700 }
|
|
4701 }
|
|
4702 }
|
|
4703
|
|
4704 ### Bowtie 2 | PAIRED-END | FASTQ
|
|
4705
|
|
4706 sub paired_end_align_fragments_to_bisulfite_genome_fastQ_bowtie2 {
|
|
4707 my ($C_to_T_infile_1,$G_to_A_infile_1,$C_to_T_infile_2,$G_to_A_infile_2) = @_;
|
|
4708 if ($directional){
|
|
4709 print "Input files are $C_to_T_infile_1 and $G_to_A_infile_2 (FastQ)\n";
|
|
4710 }
|
|
4711 else{
|
|
4712 print "Input files are $C_to_T_infile_1 and $G_to_A_infile_1 and $C_to_T_infile_2 and $G_to_A_infile_2 (FastQ)\n";
|
|
4713 }
|
|
4714
|
|
4715 ## Now starting up 4 instances of Bowtie 2 feeding in the converted sequence files and reading in the first line of the bowtie output, and storing it in the
|
|
4716 ## data structure above
|
|
4717 if ($directional){
|
|
4718 warn "Now running 2 instances of Bowtie 2 against the bisulfite genome of $genome_folder with the specified options: $bowtie_options\n\n";
|
|
4719 }
|
|
4720 else{
|
|
4721 warn "Now running 4 individual instances of Bowtie 2 against the bisulfite genome of $genome_folder with the specified options: $bowtie_options\n\n";
|
|
4722 }
|
|
4723
|
|
4724 foreach my $fh (@fhs) {
|
|
4725
|
|
4726 if ($directional){
|
|
4727 unless ($fh->{inputfile_1}){
|
|
4728 $fh->{last_seq_id} = undef;
|
|
4729 $fh->{last_line_1} = undef;
|
|
4730 $fh->{last_line_2} = undef;
|
|
4731 next;
|
|
4732 }
|
|
4733 }
|
|
4734
|
|
4735 my $bt2_options = $bowtie_options;
|
|
4736 if ($fh->{name} eq 'CTread1GAread2CTgenome' or $fh->{name} eq 'GAread1CTread2GAgenome'){
|
|
4737 $bt2_options .= ' --norc'; ### ensuring the alignments are only reported in a sensible manner
|
|
4738 }
|
|
4739 else {
|
|
4740 $bt2_options .= ' --nofw';
|
|
4741 }
|
|
4742
|
|
4743 warn "Now starting a Bowtie 2 paired-end alignment for $fh->{name} (reading in sequences from $temp_dir$fh->{inputfile_1} and $temp_dir$fh->{inputfile_2}, with the options: $bt2_options))\n";
|
|
4744 open ($fh->{fh},"$path_to_bowtie $bt2_options $fh->{bisulfiteIndex} -1 $temp_dir$fh->{inputfile_1} -2 $temp_dir$fh->{inputfile_2} |") or die "Can't open pipe to bowtie: $!";
|
|
4745
|
|
4746 ### Bowtie 2 outputs out SAM format, so we need to skip everything until the first sequence
|
|
4747 while (1){
|
|
4748 $_ = $fh->{fh}->getline();
|
|
4749 if ($_) {
|
|
4750 last unless ($_ =~ /^\@/); # SAM headers start with @
|
|
4751 }
|
|
4752 else{
|
|
4753 last; # no alignment output
|
|
4754 }
|
|
4755 }
|
|
4756
|
|
4757 my $line_1 = $_;
|
|
4758 my $line_2 = $fh->{fh}->getline();
|
|
4759
|
|
4760 # if Bowtie produces an alignment we store the first line of the output
|
|
4761 if ($line_1 and $line_2) {
|
|
4762 chomp $line_1;
|
|
4763 chomp $line_2;
|
|
4764 ### Bowtie always reports the alignment with the smaller chromosomal position first. This can be either sequence 1 or sequence 2.
|
|
4765 ### We will thus identify which sequence was read 1 and store this ID as last_seq_id
|
|
4766
|
|
4767 my $id_1 = (split(/\t/,$line_1))[0]; # this is the first element of the first bowtie output line (= the sequence identifier)
|
|
4768 my $id_2 = (split(/\t/,$line_2))[0]; # this is the first element of the second bowtie output line
|
|
4769
|
|
4770 if ($id_1 =~ s/\/1$//){ # removing the read 1 tag if present (remember that Bowtie2 clips off /1 or /2 line endings itself, so we added /1/1 or /2/2 to start with
|
|
4771 $fh->{last_seq_id} = $id_1;
|
|
4772 }
|
|
4773 elsif ($id_2 =~ s/\/1$//){ # removing the read 1 tag if present
|
|
4774 $fh->{last_seq_id} = $id_2;
|
|
4775 }
|
|
4776 else{
|
|
4777 die "Either the first or the second id need to be read 1! ID1 was: $id_1; ID2 was: $id_2\n";
|
|
4778 }
|
|
4779
|
|
4780 $fh->{last_line_1} = $line_1; # this contains read 1 or read 2
|
|
4781 $fh->{last_line_2} = $line_2; # this contains read 1 or read 2
|
|
4782 warn "Found first alignment:\n$fh->{last_line_1}\n$fh->{last_line_2}\n";
|
|
4783 }
|
|
4784
|
|
4785 # otherwise we just initialise last_seq_id and last_lines as undefined
|
|
4786 else {
|
|
4787 print "Found no alignment, assigning undef to last_seq_id and last_lines\n";
|
|
4788 $fh->{last_seq_id} = undef;
|
|
4789 $fh->{last_line_1} = undef;
|
|
4790 $fh->{last_line_2} = undef;
|
|
4791 }
|
|
4792 }
|
|
4793 }
|
|
4794
|
|
4795 #####################################################################################################################################################
|
|
4796
|
|
4797 ### Bowtie 1 (default) | SINGLE-END | FASTA
|
|
4798 sub single_end_align_fragments_to_bisulfite_genome_fastA {
|
|
4799 my ($C_to_T_infile,$G_to_A_infile) = @_;
|
|
4800 if ($directional){
|
|
4801 print "Input file is $C_to_T_infile (FastA)\n";
|
|
4802 }
|
|
4803 else{
|
|
4804 print "Input files are $C_to_T_infile and $G_to_A_infile (FastA)\n";
|
|
4805 }
|
|
4806
|
|
4807 ## Now starting up to 4 instances of Bowtie feeding in the converted sequence files and reading in the first line of the bowtie output, and storing it in
|
|
4808 ## data structure above
|
|
4809 if ($directional){
|
|
4810 warn "Now running 2 instances of Bowtie against the bisulfite genome of $genome_folder with the specified options: $bowtie_options\n\n";
|
|
4811 }
|
|
4812 else{
|
|
4813 warn "Now running 4 individual instances of Bowtie against the bisulfite genome of $genome_folder with the specified options: $bowtie_options\n\n";
|
|
4814 }
|
|
4815
|
|
4816 foreach my $fh (@fhs) {
|
|
4817
|
|
4818 my $bt_options = $bowtie_options;
|
|
4819 if ($fh->{name} eq 'CTreadCTgenome' or $fh->{name} eq 'GAreadGAgenome'){
|
|
4820 $bt_options .= ' --norc'; ### ensuring the alignments are only reported in a sensible manner
|
|
4821 }
|
|
4822 else {
|
|
4823 $bt_options .= ' --nofw';
|
|
4824 }
|
|
4825
|
|
4826 warn "Now starting the Bowtie aligner for $fh->{name} (reading in sequences from $temp_dir$fh->{inputfile} with options: $bt_options)\n";
|
|
4827 open ($fh->{fh},"$path_to_bowtie $bt_options $fh->{bisulfiteIndex} $temp_dir$fh->{inputfile} |") or die "Can't open pipe to bowtie: $!";
|
|
4828
|
|
4829 # if Bowtie produces an alignment we store the first line of the output
|
|
4830 $_ = $fh->{fh}->getline();
|
|
4831 if ($_) {
|
|
4832 chomp;
|
|
4833 my $id = (split(/\t/))[0]; # this is the first element of the bowtie output (= the sequence identifier)
|
|
4834 $fh->{last_seq_id} = $id;
|
|
4835 $fh->{last_line} = $_;
|
|
4836 warn "Found first alignment:\t$fh->{last_line}\n";
|
|
4837 }
|
|
4838 # otherwise we just initialise last_seq_id and last_line as undefined
|
|
4839 else {
|
|
4840 print "Found no alignment, assigning undef to last_seq_id and last_line\n";
|
|
4841 $fh->{last_seq_id} = undef;
|
|
4842 $fh->{last_line} = undef;
|
|
4843 }
|
|
4844 }
|
|
4845 }
|
|
4846
|
|
4847 ### Bowtie 2 | SINGLE-END | FASTA
|
|
4848 sub single_end_align_fragments_to_bisulfite_genome_fastA_bowtie2 {
|
|
4849 my ($C_to_T_infile,$G_to_A_infile) = @_;
|
|
4850 if ($directional){
|
|
4851 print "Input file is $C_to_T_infile (FastA)\n";
|
|
4852 }
|
|
4853 else{
|
|
4854 print "Input files are $C_to_T_infile and $G_to_A_infile (FastA)\n";
|
|
4855 }
|
|
4856
|
|
4857 ## Now starting up to 4 instances of Bowtie feeding in the converted sequence files and reading in the first line of the bowtie output, and storing it in
|
|
4858 ## data structure above
|
|
4859 if ($directional){
|
|
4860 warn "Now running 2 instances of Bowtie 2 against the bisulfite genome of $genome_folder with the specified options: $bowtie_options\n\n";
|
|
4861 }
|
|
4862 else{
|
|
4863 warn "Now running 4 individual instances of Bowtie 2 against the bisulfite genome of $genome_folder with the specified options: $bowtie_options\n\n";
|
|
4864 }
|
|
4865
|
|
4866 foreach my $fh (@fhs) {
|
|
4867
|
|
4868 my $bt2_options = $bowtie_options;
|
|
4869 if ($fh->{name} eq 'CTreadCTgenome' or $fh->{name} eq 'GAreadGAgenome'){
|
|
4870 $bt2_options .= ' --norc'; ### ensuring the alignments are only reported in a sensible manner
|
|
4871 }
|
|
4872 else {
|
|
4873 $bt2_options .= ' --nofw';
|
|
4874 }
|
|
4875
|
|
4876 warn "Now starting the Bowtie 2 aligner for $fh->{name} (reading in sequences from $temp_dir$fh->{inputfile} with options: $bt2_options)\n";
|
|
4877 open ($fh->{fh},"$path_to_bowtie $bt2_options $fh->{bisulfiteIndex} -U $temp_dir$fh->{inputfile} |") or die "Can't open pipe to bowtie: $!";
|
|
4878
|
|
4879 ### Bowtie 2 outputs out SAM format, so we need to skip everything until the first sequence
|
|
4880 while (1){
|
|
4881 $_ = $fh->{fh}->getline();
|
|
4882 if ($_) {
|
|
4883 last unless ($_ =~ /^\@/); # SAM headers start with @
|
|
4884 }
|
|
4885 else{
|
|
4886 last; # no alignment output
|
|
4887 }
|
|
4888 }
|
|
4889
|
|
4890 # Bowtie 2 outputs a result line even for sequences without any alignments. We thus store the first line of the output
|
|
4891 if ($_) {
|
|
4892 chomp;
|
|
4893 my $id = (split(/\t/))[0]; # this is the first element of the Bowtie output (= the sequence identifier)
|
|
4894 $fh->{last_seq_id} = $id;
|
|
4895 $fh->{last_line} = $_;
|
|
4896 warn "Found first alignment:\t$fh->{last_line}\n";
|
|
4897 }
|
|
4898 # otherwise we just initialise last_seq_id and last_line as undefinded. This should only happen at the end of a file for Bowtie 2 output
|
|
4899 else {
|
|
4900 print "Found no alignment, assigning undef to last_seq_id and last_line\n";
|
|
4901 $fh->{last_seq_id} = undef;
|
|
4902 $fh->{last_line} = undef;
|
|
4903 }
|
|
4904 }
|
|
4905 }
|
|
4906
|
|
4907
|
|
4908 ### Bowtie 1 (default) | SINGLE-END | FASTQ
|
|
4909 sub single_end_align_fragments_to_bisulfite_genome_fastQ {
|
|
4910 my ($C_to_T_infile,$G_to_A_infile) = @_;
|
|
4911 if ($directional){
|
|
4912 print "Input file is $C_to_T_infile (FastQ)\n";
|
|
4913 }
|
|
4914 else{
|
|
4915 print "Input files are $C_to_T_infile and $G_to_A_infile (FastQ)\n";
|
|
4916 }
|
|
4917
|
|
4918 ## Now starting up to 4 instances of Bowtie feeding in the converted sequence files and reading in the first line of the bowtie output, and storing it in
|
|
4919 ## the data structure above
|
|
4920 if ($directional){
|
|
4921 warn "Now running 2 instances of Bowtie against the bisulfite genome of $genome_folder with the specified options: $bowtie_options\n\n";
|
|
4922 }
|
|
4923 else{
|
|
4924 warn "Now running 4 individual instances of Bowtie against the bisulfite genome of $genome_folder with the specified options: $bowtie_options\n\n";
|
|
4925 }
|
|
4926
|
|
4927 foreach my $fh (@fhs) {
|
|
4928 my $bt_options = $bowtie_options;
|
|
4929 if ($fh->{name} eq 'CTreadCTgenome' or $fh->{name} eq 'GAreadGAgenome'){
|
|
4930 $bt_options .= ' --norc'; ### ensuring the alignments are only reported in a sensible manner
|
|
4931 }
|
|
4932 else {
|
|
4933 $bt_options .= ' --nofw';
|
|
4934 }
|
|
4935
|
|
4936 warn "Now starting the Bowtie aligner for $fh->{name} (reading in sequences from $temp_dir$fh->{inputfile} with options: $bt_options)\n";
|
|
4937 open ($fh->{fh},"$path_to_bowtie $bowtie_options $fh->{bisulfiteIndex} $temp_dir$fh->{inputfile} |") or die "Can't open pipe to bowtie: $!";
|
|
4938
|
|
4939 # if Bowtie produces an alignment we store the first line of the output
|
|
4940 $_ = $fh->{fh}->getline();
|
|
4941 if ($_) {
|
|
4942 chomp;
|
|
4943 my $id = (split(/\t/))[0]; # this is the first element of the Bowtie output (= the sequence identifier)
|
|
4944 $fh->{last_seq_id} = $id;
|
|
4945 $fh->{last_line} = $_;
|
|
4946 warn "Found first alignment:\t$fh->{last_line}\n";
|
|
4947 }
|
|
4948 # otherwise we just initialise last_seq_id and last_line as undefined
|
|
4949 else {
|
|
4950 print "Found no alignment, assigning undef to last_seq_id and last_line\n";
|
|
4951 $fh->{last_seq_id} = undef;
|
|
4952 $fh->{last_line} = undef;
|
|
4953 }
|
|
4954 }
|
|
4955 }
|
|
4956
|
|
4957 ### Bowtie 2 | SINGLE-END | FASTQ
|
|
4958 sub single_end_align_fragments_to_bisulfite_genome_fastQ_bowtie2 {
|
|
4959 my ($C_to_T_infile,$G_to_A_infile) = @_;
|
|
4960 if ($directional){
|
|
4961 print "Input file is $C_to_T_infile (FastQ)\n\n";
|
|
4962 }
|
|
4963 else{
|
|
4964 print "Input files are $C_to_T_infile and $G_to_A_infile (FastQ)\n\n";
|
|
4965 }
|
|
4966
|
|
4967 ## Now starting up to 4 instances of Bowtie 2 feeding in the converted sequence files and reading in the first line of the bowtie output, and storing it in
|
|
4968 ## the data structure above
|
|
4969 if ($directional){
|
|
4970 warn "Now running 2 instances of Bowtie 2 against the bisulfite genome of $genome_folder with the specified options: $bowtie_options\n\n";
|
|
4971 }
|
|
4972 else{
|
|
4973 warn "Now running 4 individual instances of Bowtie 2 against the bisulfite genome of $genome_folder with the specified options: $bowtie_options\n\n";
|
|
4974 }
|
|
4975
|
|
4976 foreach my $fh (@fhs) {
|
|
4977 my $bt2_options = $bowtie_options;
|
|
4978 if ($fh->{name} eq 'CTreadCTgenome' or $fh->{name} eq 'GAreadGAgenome'){
|
|
4979 $bt2_options .= ' --norc'; ### ensuring the alignments are only reported in a sensible manner
|
|
4980 }
|
|
4981 else {
|
|
4982 $bt2_options .= ' --nofw';
|
|
4983 }
|
|
4984 warn "Now starting the Bowtie 2 aligner for $fh->{name} (reading in sequences from $temp_dir$fh->{inputfile} with options $bt2_options)\n";
|
|
4985 warn "Using Bowtie 2 index: $fh->{bisulfiteIndex}\n\n";
|
|
4986
|
|
4987 open ($fh->{fh},"$path_to_bowtie $bt2_options $fh->{bisulfiteIndex} -U $temp_dir$fh->{inputfile} |") or die "Can't open pipe to bowtie: $!";
|
|
4988 ### Bowtie 2 outputs out SAM format, so we need to skip everything until the first sequence
|
|
4989 while (1){
|
|
4990 $_ = $fh->{fh}->getline();
|
|
4991 if ($_) {
|
|
4992 last unless ($_ =~ /^\@/); # SAM headers start with @
|
|
4993 }
|
|
4994 else {
|
|
4995 last;
|
|
4996 }
|
|
4997 }
|
|
4998
|
|
4999 # Bowtie 2 outputs a result line even for sequences without any alignments. We thus store the first line of the output
|
|
5000 if ($_) {
|
|
5001 chomp;
|
|
5002 my $id = (split(/\t/))[0]; # this is the first element of the Bowtie 2 output (= the sequence identifier)
|
|
5003 $fh->{last_seq_id} = $id;
|
|
5004 $fh->{last_line} = $_;
|
|
5005 warn "Found first alignment:\t$fh->{last_line}\n";
|
|
5006 }
|
|
5007 # otherwise we just initialise last_seq_id and last_line as undefined. This should only happen at the end of a file for Bowtie 2 output
|
|
5008 else {
|
|
5009 print "Found no alignment, assigning undef to last_seq_id and last_line\n";
|
|
5010 $fh->{last_seq_id} = undef;
|
|
5011 $fh->{last_line} = undef;
|
|
5012 }
|
|
5013 }
|
|
5014 }
|
|
5015
|
|
5016 ###########################################################################################################################################
|
|
5017
|
|
5018 sub reset_counters_and_fhs{
|
|
5019 my $filename = shift;
|
|
5020 %counting=(
|
|
5021 total_meCHH_count => 0,
|
|
5022 total_meCHG_count => 0,
|
|
5023 total_meCpG_count => 0,
|
|
5024 total_unmethylated_CHH_count => 0,
|
|
5025 total_unmethylated_CHG_count => 0,
|
|
5026 total_unmethylated_CpG_count => 0,
|
|
5027 sequences_count => 0,
|
|
5028 no_single_alignment_found => 0,
|
|
5029 unsuitable_sequence_count => 0,
|
|
5030 genomic_sequence_could_not_be_extracted_count => 0,
|
|
5031 unique_best_alignment_count => 0,
|
|
5032 low_complexity_alignments_overruled_count => 0,
|
|
5033 CT_CT_count => 0, #(CT read/CT genome, original top strand)
|
|
5034 CT_GA_count => 0, #(CT read/GA genome, original bottom strand)
|
|
5035 GA_CT_count => 0, #(GA read/CT genome, complementary to original top strand)
|
|
5036 GA_GA_count => 0, #(GA read/GA genome, complementary to original bottom strand)
|
|
5037 CT_GA_CT_count => 0, #(CT read1/GA read2/CT genome, original top strand)
|
|
5038 GA_CT_GA_count => 0, #(GA read1/CT read2/GA genome, complementary to original bottom strand)
|
|
5039 GA_CT_CT_count => 0, #(GA read1/CT read2/CT genome, complementary to original top strand)
|
|
5040 CT_GA_GA_count => 0, #(CT read1/GA read2/GA genome, original bottom strand)
|
|
5041 alignments_rejected_count => 0, # only relevant if --directional was specified
|
|
5042 );
|
|
5043
|
|
5044 if ($directional){
|
|
5045 if ($filename =~ ','){ # paired-end files
|
|
5046 @fhs=(
|
|
5047 { name => 'CTreadCTgenome',
|
|
5048 strand_identity => 'con ori forward',
|
|
5049 bisulfiteIndex => $CT_index_basename,
|
|
5050 seen => 0,
|
|
5051 wrong_strand => 0,
|
|
5052 },
|
|
5053 { name => 'CTreadGAgenome',
|
|
5054 strand_identity => 'con ori reverse',
|
|
5055 bisulfiteIndex => $GA_index_basename,
|
|
5056 seen => 0,
|
|
5057 wrong_strand => 0,
|
|
5058 },
|
|
5059 { name => 'GAreadCTgenome',
|
|
5060 strand_identity => 'compl ori con forward',
|
|
5061 bisulfiteIndex => $CT_index_basename,
|
|
5062 seen => 0,
|
|
5063 wrong_strand => 0,
|
|
5064 },
|
|
5065 { name => 'GAreadGAgenome',
|
|
5066 strand_identity => 'compl ori con reverse',
|
|
5067 bisulfiteIndex => $GA_index_basename,
|
|
5068 seen => 0,
|
|
5069 wrong_strand => 0,
|
|
5070 },
|
|
5071 );
|
|
5072 }
|
|
5073 else{ # single-end files
|
|
5074 @fhs=(
|
|
5075 { name => 'CTreadCTgenome',
|
|
5076 strand_identity => 'con ori forward',
|
|
5077 bisulfiteIndex => $CT_index_basename,
|
|
5078 seen => 0,
|
|
5079 wrong_strand => 0,
|
|
5080 },
|
|
5081 { name => 'CTreadGAgenome',
|
|
5082 strand_identity => 'con ori reverse',
|
|
5083 bisulfiteIndex => $GA_index_basename,
|
|
5084 seen => 0,
|
|
5085 wrong_strand => 0,
|
|
5086 },
|
|
5087 );
|
|
5088 }
|
|
5089 }
|
|
5090 else{
|
|
5091 @fhs=(
|
|
5092 { name => 'CTreadCTgenome',
|
|
5093 strand_identity => 'con ori forward',
|
|
5094 bisulfiteIndex => $CT_index_basename,
|
|
5095 seen => 0,
|
|
5096 wrong_strand => 0,
|
|
5097 },
|
|
5098 { name => 'CTreadGAgenome',
|
|
5099 strand_identity => 'con ori reverse',
|
|
5100 bisulfiteIndex => $GA_index_basename,
|
|
5101 seen => 0,
|
|
5102 wrong_strand => 0,
|
|
5103 },
|
|
5104 { name => 'GAreadCTgenome',
|
|
5105 strand_identity => 'compl ori con forward',
|
|
5106 bisulfiteIndex => $CT_index_basename,
|
|
5107 seen => 0,
|
|
5108 wrong_strand => 0,
|
|
5109 },
|
|
5110 { name => 'GAreadGAgenome',
|
|
5111 strand_identity => 'compl ori con reverse',
|
|
5112 bisulfiteIndex => $GA_index_basename,
|
|
5113 seen => 0,
|
|
5114 wrong_strand => 0,
|
|
5115 },
|
|
5116 );
|
|
5117 }
|
|
5118 }
|
|
5119
|
|
5120
|
|
5121 sub process_command_line{
|
|
5122 my @bowtie_options;
|
|
5123 my $help;
|
|
5124 my $mates1;
|
|
5125 my $mates2;
|
|
5126 my $path_to_bowtie;
|
|
5127 my $fastq;
|
|
5128 my $fasta;
|
|
5129 my $skip;
|
|
5130 my $qupto;
|
|
5131 my $phred64;
|
|
5132 my $phred33;
|
|
5133 my $solexa;
|
|
5134 my $mismatches;
|
|
5135 my $seed_length;
|
|
5136 my $best;
|
|
5137 my $sequence_format;
|
|
5138 my $version;
|
|
5139 my $quiet;
|
|
5140 my $chunk;
|
|
5141 my $non_directional;
|
|
5142 my $ceiling;
|
|
5143 my $maxins;
|
|
5144 my $minins;
|
|
5145 my $unmapped;
|
|
5146 my $multi_map;
|
|
5147 my $output_dir;
|
|
5148 my $bowtie2;
|
|
5149 my $vanilla;
|
|
5150 my $sam_no_hd;
|
|
5151 my $seed_extension_fails;
|
|
5152 my $reseed_repetitive_seeds;
|
|
5153 my $most_valid_alignments;
|
|
5154 my $score_min;
|
|
5155 my $parallel;
|
|
5156 my $temp_dir;
|
|
5157 my $rdg;
|
|
5158 my $rfg;
|
|
5159
|
|
5160 my $command_line = GetOptions ('help|man' => \$help,
|
|
5161 '1=s' => \$mates1,
|
|
5162 '2=s' => \$mates2,
|
|
5163 'path_to_bowtie=s' => \$path_to_bowtie,
|
|
5164 'f|fasta' => \$fasta,
|
|
5165 'q|fastq' => \$fastq,
|
|
5166 's|skip=i' => \$skip,
|
|
5167 'u|upto=i' => \$qupto,
|
|
5168 'phred33-quals' => \$phred33,
|
|
5169 'phred64-quals|solexa1' => \$phred64,
|
|
5170 'solexa-quals' => \$solexa,
|
|
5171 'n|seedmms=i' => \$mismatches,
|
|
5172 'l|seedlen=i' => \$seed_length,
|
|
5173 'no_best' => \$best,
|
|
5174 'version' => \$version,
|
|
5175 'quiet' => \$quiet,
|
|
5176 'chunkmbs=i' => \$chunk,
|
|
5177 'non_directional' => \$non_directional,
|
|
5178 'I|minins=i' => \$minins,
|
|
5179 'X|maxins=i' => \$maxins,
|
|
5180 'e|maqerr=i' => \$ceiling,
|
|
5181 'un|unmapped' => \$unmapped,
|
|
5182 'ambiguous' => \$multi_map,
|
|
5183 'o|output_dir=s' => \$output_dir,
|
|
5184 'bowtie2' => \$bowtie2,
|
|
5185 'vanilla' => \$vanilla,
|
|
5186 'sam-no-hd' => \$sam_no_hd,
|
|
5187 'D=i' => \$seed_extension_fails,
|
|
5188 'R=i' => \$reseed_repetitive_seeds,
|
|
5189 'score_min=s' => \$score_min,
|
|
5190 'most_valid_alignments=i' => \$most_valid_alignments,
|
|
5191 'p=i' => \$parallel,
|
|
5192 'temp_dir=s' => \$temp_dir,
|
|
5193 'rdg=s' => \$rdg,
|
|
5194 'rfg=s' => \$rfg,
|
|
5195 );
|
|
5196
|
|
5197
|
|
5198 ### EXIT ON ERROR if there were errors with any of the supplied options
|
|
5199 unless ($command_line){
|
|
5200 die "Please respecify command line options\n";
|
|
5201 }
|
|
5202 ### HELPFILE
|
|
5203 if ($help){
|
|
5204 print_helpfile();
|
|
5205 exit;
|
|
5206 }
|
|
5207 if ($version){
|
|
5208 print << "VERSION";
|
|
5209
|
|
5210
|
|
5211 Bismark - Bisulfite Mapper and Methylation Caller.
|
|
5212
|
|
5213 Bismark Version: $bismark_version Copyright 2010-12 Felix Krueger, Babraham Bioinformatics
|
|
5214 www.bioinformatics.babraham.ac.uk/projects/
|
|
5215
|
|
5216
|
|
5217 VERSION
|
|
5218 exit;
|
|
5219 }
|
|
5220
|
|
5221
|
|
5222 ##########################
|
|
5223 ### PROCESSING OPTIONS ###
|
|
5224 ##########################
|
|
5225
|
|
5226 unless ($bowtie2){
|
|
5227 $bowtie2 = 0;
|
|
5228 }
|
|
5229 unless ($sam_no_hd){
|
|
5230 $sam_no_hd =0;
|
|
5231 }
|
|
5232
|
|
5233 ### PATH TO BOWTIE
|
|
5234 ### if a special path to Bowtie 1/2 was specified we will use that one, otherwise it is assumed that Bowtie 1/2 is in the PATH
|
|
5235 if ($path_to_bowtie){
|
|
5236 unless ($path_to_bowtie =~ /\/$/){
|
|
5237 $path_to_bowtie =~ s/$/\//;
|
|
5238 }
|
|
5239 if (-d $path_to_bowtie){
|
|
5240 if ($bowtie2){
|
|
5241 $path_to_bowtie = "${path_to_bowtie}bowtie2";
|
|
5242 }
|
|
5243 else{
|
|
5244 $path_to_bowtie = "${path_to_bowtie}bowtie";
|
|
5245 }
|
|
5246 }
|
|
5247 else{
|
|
5248 die "The path to bowtie provided ($path_to_bowtie) is invalid (not a directory)!\n";
|
|
5249 }
|
|
5250 }
|
|
5251 else{
|
|
5252 if ($bowtie2){
|
|
5253 $path_to_bowtie = 'bowtie2';
|
|
5254 warn "Path to Bowtie 2 specified as: $path_to_bowtie\n"; }
|
|
5255 else{
|
|
5256 $path_to_bowtie = 'bowtie';
|
|
5257 warn "Path to Bowtie specified as: $path_to_bowtie\n";
|
|
5258 }
|
|
5259 }
|
|
5260
|
|
5261 ####################################
|
|
5262 ### PROCESSING ARGUMENTS
|
|
5263
|
|
5264 ### GENOME FOLDER
|
|
5265 my $genome_folder = shift @ARGV; # mandatory
|
|
5266 unless ($genome_folder){
|
|
5267 warn "Genome folder was not specified!\n";
|
|
5268 print_helpfile();
|
|
5269 exit;
|
|
5270 }
|
|
5271
|
|
5272 ### checking that the genome folder, all subfolders and the required bowtie index files exist
|
|
5273 unless ($genome_folder =~/\/$/){
|
|
5274 $genome_folder =~ s/$/\//;
|
|
5275 }
|
|
5276
|
|
5277 if (chdir $genome_folder){
|
|
5278 my $absolute_genome_folder = getcwd; ## making the genome folder path absolute
|
|
5279 unless ($absolute_genome_folder =~/\/$/){
|
|
5280 $absolute_genome_folder =~ s/$/\//;
|
|
5281 }
|
|
5282 warn "Reference genome folder provided is $genome_folder\t(absolute path is '$absolute_genome_folder)'\n";
|
|
5283 $genome_folder = $absolute_genome_folder;
|
|
5284 }
|
|
5285 else{
|
|
5286 die "Failed to move to $genome_folder: $!\nUSAGE: Bismark.pl [options] <genome_folder> {-1 <mates1> -2 <mates2> | <singles>} [<hits>] (--help for more details)\n";
|
|
5287 }
|
|
5288
|
|
5289 my $CT_dir = "${genome_folder}Bisulfite_Genome/CT_conversion/";
|
|
5290 my $GA_dir = "${genome_folder}Bisulfite_Genome/GA_conversion/";
|
|
5291
|
|
5292 if ($bowtie2){ ### Bowtie 2 (new)
|
|
5293 ### checking the integrity of $CT_dir
|
|
5294 chdir $CT_dir or die "Failed to move to directory $CT_dir: $!\n";
|
|
5295 my @CT_bowtie_index = ('BS_CT.1.bt2','BS_CT.2.bt2','BS_CT.3.bt2','BS_CT.4.bt2','BS_CT.rev.1.bt2','BS_CT.rev.2.bt2');
|
|
5296 foreach my $file(@CT_bowtie_index){
|
|
5297 unless (-f $file){
|
|
5298 die "The Bowtie 2 index of the C->T converted genome seems to be faulty ($file). Please run the bismark_genome_preparation before running Bismark.\n";
|
|
5299 }
|
|
5300 }
|
|
5301 ### checking the integrity of $GA_dir
|
|
5302 chdir $GA_dir or die "Failed to move to directory $GA_dir: $!\n";
|
|
5303 my @GA_bowtie_index = ('BS_GA.1.bt2','BS_GA.2.bt2','BS_GA.3.bt2','BS_GA.4.bt2','BS_GA.rev.1.bt2','BS_GA.rev.2.bt2');
|
|
5304 foreach my $file(@GA_bowtie_index){
|
|
5305 unless (-f $file){
|
|
5306 die "The Bowtie 2 index of the G->A converted genome seems to be faulty ($file). Please run bismark_genome_preparation before running Bismark.\n";
|
|
5307 }
|
|
5308 }
|
|
5309 }
|
|
5310
|
|
5311 else{ ### Bowtie 1 (default)
|
|
5312 ### checking the integrity of $CT_dir
|
|
5313 chdir $CT_dir or die "Failed to move to directory $CT_dir: $!\n";
|
|
5314 my @CT_bowtie_index = ('BS_CT.1.ebwt','BS_CT.2.ebwt','BS_CT.3.ebwt','BS_CT.4.ebwt','BS_CT.rev.1.ebwt','BS_CT.rev.2.ebwt');
|
|
5315 foreach my $file(@CT_bowtie_index){
|
|
5316 unless (-f $file){
|
|
5317 die "The Bowtie index of the C->T converted genome seems to be faulty ($file). Please run bismark_genome_preparation before running Bismark.\n";
|
|
5318 }
|
|
5319 }
|
|
5320 ### checking the integrity of $GA_dir
|
|
5321 chdir $GA_dir or die "Failed to move to directory $GA_dir: $!\n";
|
|
5322 my @GA_bowtie_index = ('BS_GA.1.ebwt','BS_GA.2.ebwt','BS_GA.3.ebwt','BS_GA.4.ebwt','BS_GA.rev.1.ebwt','BS_GA.rev.2.ebwt');
|
|
5323 foreach my $file(@GA_bowtie_index){
|
|
5324 unless (-f $file){
|
|
5325 die "The Bowtie index of the G->A converted genome seems to be faulty ($file). Please run bismark_genome_preparation before running Bismark.\n";
|
|
5326 }
|
|
5327 }
|
|
5328 }
|
|
5329
|
|
5330 my $CT_index_basename = "${CT_dir}BS_CT";
|
|
5331 my $GA_index_basename = "${GA_dir}BS_GA";
|
|
5332
|
|
5333 ### INPUT OPTIONS
|
|
5334
|
|
5335 ### SEQUENCE FILE FORMAT
|
|
5336 ### exits if both fastA and FastQ were specified
|
|
5337 if ($fasta and $fastq){
|
|
5338 die "Only one sequence filetype can be specified (fastA or fastQ)\n";
|
|
5339 }
|
|
5340
|
|
5341 ### unless fastA is specified explicitely, fastQ sequence format is expected by default
|
|
5342 if ($fasta){
|
|
5343 print "FastA format specified\n";
|
|
5344 $sequence_format = 'FASTA';
|
|
5345 push @bowtie_options, '-f';
|
|
5346 }
|
|
5347 elsif ($fastq){
|
|
5348 print "FastQ format specified\n";
|
|
5349 $sequence_format = 'FASTQ';
|
|
5350 push @bowtie_options, '-q';
|
|
5351 }
|
|
5352 else{
|
|
5353 $fastq = 1;
|
|
5354 print "FastQ format assumed (by default)\n";
|
|
5355 $sequence_format = 'FASTQ';
|
|
5356 push @bowtie_options, '-q';
|
|
5357 }
|
|
5358
|
|
5359 ### SKIP
|
|
5360 if ($skip){
|
|
5361 warn "Skipping the first $skip reads from the input file\n";
|
|
5362 # push @bowtie_options,"-s $skip";
|
|
5363 }
|
|
5364
|
|
5365 ### UPTO
|
|
5366 if ($qupto){
|
|
5367 warn "Processing sequences up to read no. $qupto from the input file\n";
|
|
5368 if ($bowtie2){
|
|
5369 # push @bowtie_options,"--upto $qupto"; ## slightly changed for Bowtie 2
|
|
5370 }
|
|
5371 else{
|
|
5372 # push @bowtie_options,"--qupto $qupto";
|
|
5373 }
|
|
5374 }
|
|
5375
|
|
5376 ### QUALITY VALUES
|
|
5377 if (($phred33 and $phred64) or ($phred33 and $solexa) or ($phred64 and $solexa)){
|
|
5378 die "You can only specify one type of quality value at a time! (--phred33-quals or --phred64-quals or --solexa-quals)";
|
|
5379 }
|
|
5380 if ($phred33){ ## if nothing else is specified $phred33 will be used as default by both Bowtie 1 and 2.
|
|
5381 # Phred quality values work only when -q is specified
|
|
5382 unless ($fastq){
|
|
5383 die "Phred quality values works only when -q (FASTQ) is specified\n";
|
|
5384 }
|
|
5385 if ($bowtie2){
|
|
5386 push @bowtie_options,"--phred33";
|
|
5387 }
|
|
5388 else{
|
|
5389 push @bowtie_options,"--phred33-quals";
|
|
5390 }
|
|
5391 }
|
|
5392 if ($phred64){
|
|
5393 # Phred quality values work only when -q is specified
|
|
5394 unless ($fastq){
|
|
5395 die "Phred quality values work only when -q (FASTQ) is specified\n";
|
|
5396 }
|
|
5397 if ($bowtie2){
|
|
5398 push @bowtie_options,"--phred64";
|
|
5399 }
|
|
5400 else{
|
|
5401 push @bowtie_options,"--phred64-quals";
|
|
5402 }
|
|
5403 }
|
|
5404 else{
|
|
5405 $phred64 = 0;
|
|
5406 }
|
|
5407
|
|
5408 if ($solexa){
|
|
5409 if ($bowtie2){
|
|
5410 die "The option '--solexa-quals' is not compatible with Bowtie 2. Please respecify!\n";
|
|
5411 }
|
|
5412 # Solexa to Phred value conversion works only when -q is specified
|
|
5413 unless ($fastq){
|
|
5414 die "Conversion from Solexa to Phred quality values works only when -q (FASTQ) is specified\n";
|
|
5415 }
|
|
5416 push @bowtie_options,"--solexa-quals";
|
|
5417 }
|
|
5418 else{
|
|
5419 $solexa = 0;
|
|
5420 }
|
|
5421
|
|
5422 ### ALIGNMENT OPTIONS
|
|
5423
|
|
5424 ### MISMATCHES
|
|
5425 if (defined $mismatches){
|
|
5426 if ($bowtie2){
|
|
5427 if ($mismatches == 0 or $mismatches == 1){
|
|
5428 push @bowtie_options,"-N $mismatches";
|
|
5429 }
|
|
5430 else{
|
|
5431 die "Please set the number of multiseed mismatches for Bowtie 2 with '-N <int>' (where <int> can be 0 or 1)\n";
|
|
5432 }
|
|
5433 }
|
|
5434 else{
|
|
5435 if ($mismatches >= 0 and $mismatches <= 3){
|
|
5436 push @bowtie_options,"-n $mismatches";
|
|
5437 }
|
|
5438 else{
|
|
5439 die "Please set the number of seed mismatches for Bowtie 1 with '-n <int>' (where <int> can be 0,1,2 or 3)\n";
|
|
5440 }
|
|
5441 }
|
|
5442 }
|
|
5443 else{
|
|
5444 unless ($bowtie2){
|
|
5445 push @bowtie_options,"-n 1"; # setting -n to 1 by default (for use with Bowtie only) because it is much quicker than the default mode of -n 2
|
|
5446 }
|
|
5447 }
|
|
5448
|
|
5449 ### SEED LENGTH
|
|
5450 if (defined $seed_length){
|
|
5451 if ($bowtie2){
|
|
5452 push @bowtie_options,"-L $seed_length";
|
|
5453 }
|
|
5454 else{
|
|
5455 push @bowtie_options,"-l $seed_length";
|
|
5456 }
|
|
5457 }
|
|
5458
|
|
5459 ### MISMATCH CEILING
|
|
5460 if (defined $ceiling){
|
|
5461 die "The option '-e' is not compatible with Bowtie 2. Please respecify options\n" if ($bowtie2);
|
|
5462 push @bowtie_options,"-e $ceiling";
|
|
5463 }
|
|
5464
|
|
5465
|
|
5466 ### BOWTIE 2 EFFORT OPTIONS
|
|
5467
|
|
5468 ### CONSECUTIVE SEED EXTENSION FAILS
|
|
5469 if (defined $seed_extension_fails){
|
|
5470 die "The option '-D <int>' is only available when using Bowtie 2\n\n" unless ($bowtie2);
|
|
5471 push @bowtie_options,"-D $seed_extension_fails";
|
|
5472 }
|
|
5473
|
|
5474 ### RE-SEEDING REPETITIVE SEEDS
|
|
5475 if (defined $reseed_repetitive_seeds){
|
|
5476 die "The option '-R <int>' is only available when using Bowtie 2\n\n" unless ($bowtie2);
|
|
5477 push @bowtie_options,"-R $reseed_repetitive_seeds";
|
|
5478 }
|
|
5479
|
|
5480
|
|
5481 ### BOWTIE 2 SCORING OPTIONS
|
|
5482 if ($score_min){
|
|
5483 die "The option '--score_min <func>' is only available when using Bowtie 2\n\n" unless ($bowtie2);
|
|
5484 unless ($score_min =~ /^L,.+,.+$/){
|
|
5485 die "The option '--score_min <func>' needs to be in the format <L,value,value> . Please consult \"setting up functions\" in the Bowtie 2 manual for further information\n\n";
|
|
5486 }
|
|
5487 push @bowtie_options,"--score-min $score_min";
|
|
5488 }
|
|
5489 else{
|
|
5490 if ($bowtie2){
|
|
5491 push @bowtie_options,"--score-min L,0,-0.2"; # default setting, more stringent than normal Bowtie2
|
|
5492 }
|
|
5493 }
|
|
5494
|
|
5495 ### BOWTIE 2 READ GAP OPTIONS
|
|
5496 if ($rdg){
|
|
5497 die "The option '--rdg <int1>,<int2>' is only available when using Bowtie 2\n\n" unless ($bowtie2);
|
|
5498 unless ($rdg =~ /^.+,.+$/){
|
|
5499 die "The option '--rdg <int1>,<int2>' needs to be in the format <integer,integer> . Please consult \"setting up functions\" in the Bowtie 2 manual for further information\n\n";
|
|
5500 }
|
|
5501 push @bowtie_options,"--rdg $rdg";
|
|
5502 }
|
|
5503
|
|
5504 ### BOWTIE 2 REFERENCE GAP OPTIONS
|
|
5505 if ($rfg){
|
|
5506 die "The option '--rfg <int1>,<int2>' is only available when using Bowtie 2\n\n" unless ($bowtie2);
|
|
5507 unless ($rfg =~ /^.+,.+$/){
|
|
5508 die "The option '--rfg <int1>,<int2>' needs to be in the format <integer,integer> . Please consult \"setting up functions\" in the Bowtie 2 manual for further information\n\n";
|
|
5509 }
|
|
5510 push @bowtie_options,"--rfg $rfg";
|
|
5511 }
|
|
5512
|
|
5513
|
|
5514
|
|
5515 ### BOWTIE 2 PARALLELIZATION OPTIONS
|
|
5516 if (defined $parallel){
|
|
5517 die "The parallelization switch '-p' only works for Bowtie 2. Please respecify!" unless ($bowtie2);
|
|
5518 }
|
|
5519 if ($bowtie2){
|
|
5520 if ($parallel){
|
|
5521 die "Please select a value for -p of 2 or more!\n" unless ($parallel > 1);
|
|
5522 push @bowtie_options,"-p $parallel";
|
|
5523 push @bowtie_options,'--reorder'; ## re-orders the bowtie 2 output so that it does match the input files. This is abolutely required for parallelization to work.
|
|
5524 print "Each Bowtie 2 instance is going to be run with $parallel threads. Please monitor performance closely and tune down if needed!\n";
|
|
5525 sleep (2);
|
|
5526 }
|
|
5527 }
|
|
5528
|
|
5529 ### REPORTING OPTIONS
|
|
5530
|
|
5531 if ($bowtie2){
|
|
5532 push @bowtie_options,'--ignore-quals'; ## All mismatches will receive penalty for mismatches as if they were of high quality, which is 6 by default
|
|
5533
|
|
5534 ### Option -M is deprecated since Bowtie 2 version 2.0.0 beta7. I'll leave this option commented out for a while
|
|
5535 if(defined $most_valid_alignments){
|
|
5536
|
|
5537 warn "\nThe option -M is now deprecated (as of Bowtie 2 version 2.0.0 beta7). What used to be called -M mode is still the default mode. Use the -D and -R options to adjust the effort expended to find valid alignments.\n\n";
|
|
5538 # push @bowtie_options,"-M $most_valid_alignments";sleep (5);
|
|
5539 }
|
|
5540 # else{
|
|
5541 # push @bowtie_options,'-M 10'; # the default behavior for Bowtie 2 is to report (and sort) up to 500 alignments for a given sequence
|
|
5542 # }
|
|
5543 }
|
|
5544 else{ # Because of the way Bismark works we will always use the reporting option -k 2 (report up to 2 valid alignments) for Bowtie 1
|
|
5545 push @bowtie_options,'-k 2';
|
|
5546 }
|
|
5547
|
|
5548 ### --BEST
|
|
5549 if ($bowtie2){
|
|
5550 if ($best){ # Bowtie 2 does away with the concept of --best, so one can also not select --no-best when Bowtie 2 is to be used
|
|
5551 die "The option '--no-best' is not compatible with Bowtie 2. Please respecify options\n";
|
|
5552 }
|
|
5553 }
|
|
5554 else{
|
|
5555 # --best is the default option for Bowtie 1, specifying --no-best can turn it off (e.g. to speed up alignment process)
|
|
5556 unless ($best){
|
|
5557 push @bowtie_options,'--best';
|
|
5558 }
|
|
5559 }
|
|
5560
|
|
5561 ### VANILLA BISMARK (BOWTIE 1) OUTPUT
|
|
5562 if ($vanilla){
|
|
5563 if ($bowtie2){
|
|
5564 die "The options --bowtie2 and the --vanilla are not compatible. Please respecify!\n\n";
|
|
5565 }
|
|
5566 }
|
|
5567 else{
|
|
5568 $vanilla = 0;
|
|
5569 }
|
|
5570
|
|
5571 ### PAIRED-END MAPPING
|
|
5572 if ($mates1){
|
|
5573 my @mates1 = (split (/,/,$mates1));
|
|
5574 die "Paired-end mapping requires the format: -1 <mates1> -2 <mates2>, please respecify!\n" unless ($mates2);
|
|
5575 my @mates2 = (split(/,/,$mates2));
|
|
5576 unless (scalar @mates1 == scalar @mates2){
|
|
5577 die "Paired-end mapping requires the same amounnt of mate1 and mate2 files, please respecify! (format: -1 <mates1> -2 <mates2>)\n";
|
|
5578 }
|
|
5579 while (1){
|
|
5580 my $mate1 = shift @mates1;
|
|
5581 my $mate2 = shift @mates2;
|
|
5582 last unless ($mate1 and $mate2);
|
|
5583 push @filenames,"$mate1,$mate2";
|
|
5584 }
|
|
5585 if ($bowtie2){
|
|
5586 push @bowtie_options,'--no-mixed'; ## By default Bowtie 2 is not looking for single-end alignments if it can't find concordant or discordant alignments
|
|
5587 push @bowtie_options,'--no-discordant';## By default Bowtie 2 is not looking for discordant alignments if it can't find concordant ones
|
|
5588 }
|
|
5589 }
|
|
5590 elsif ($mates2){
|
|
5591 die "Paired-end mapping requires the format: -1 <mates1> -2 <mates2>, please respecify!\n";
|
|
5592 }
|
|
5593
|
|
5594 ### SINGLE-END MAPPING
|
|
5595 # Single-end mapping will be performed if no mate pairs for paired-end mapping have been specified
|
|
5596 my $singles;
|
|
5597 unless ($mates1 and $mates2){
|
|
5598 $singles = join (',',@ARGV);
|
|
5599 unless ($singles){
|
|
5600 die "\nNo filename supplied! Please specify one or more files for single-end Bismark mapping!\n";
|
|
5601 }
|
|
5602 $singles =~ s/\s/,/g;
|
|
5603 @filenames = (split(/,/,$singles));
|
|
5604 warn "\nFiles to be analysed:\n";
|
|
5605 warn "@filenames\n\n";
|
|
5606 sleep (3);
|
|
5607 }
|
|
5608
|
|
5609 ### MININUM INSERT SIZE (PAIRED-END ONLY)
|
|
5610 if (defined $minins){
|
|
5611 die "-I/--minins can only be used for paired-end mapping!\n\n" if ($singles);
|
|
5612 push @bowtie_options,"--minins $minins";
|
|
5613 }
|
|
5614
|
|
5615 ### MAXIMUM INSERT SIZE (PAIRED-END ONLY)
|
|
5616 if (defined $maxins){
|
|
5617 die "-X/--maxins can only be used for paired-end mapping!\n\n" if ($singles);
|
|
5618 push @bowtie_options,"--maxins $maxins";
|
|
5619 }
|
|
5620 else{
|
|
5621 unless ($singles){
|
|
5622 push @bowtie_options,'--maxins 500';
|
|
5623 }
|
|
5624 }
|
|
5625
|
|
5626 ### QUIET prints nothing besides alignments (suppresses warnings)
|
|
5627 if ($quiet){
|
|
5628 push @bowtie_options,'--quiet';
|
|
5629 }
|
|
5630
|
|
5631 ### CHUNKMBS needed to be increased to avoid memory exhaustion warnings for Bowtie 1, particularly for --best (and paired-end) alignments
|
|
5632 unless ($bowtie2){ # Bowtie 2 does not have a chunkmbs option
|
|
5633 if (defined $chunk){
|
|
5634 push @bowtie_options,"--chunkmbs $chunk";
|
|
5635 }
|
|
5636 else{
|
|
5637 push @bowtie_options,'--chunkmbs 512'; ## setting the default to 512MB (up from 64 default)
|
|
5638 }
|
|
5639 }
|
|
5640
|
|
5641
|
|
5642 ### SUMMARY OF ALL BOWTIE OPTIONS
|
|
5643 my $bowtie_options = join (' ',@bowtie_options);
|
|
5644
|
|
5645
|
|
5646 ### STRAND-SPECIFIC LIBRARIES
|
|
5647 my $directional;
|
|
5648 if ($non_directional){
|
|
5649 print "Library was specified to be not strand-specific (non-directional), therefore alignments to all four possible bisulfite strands (OT, CTOT, OB and CTOB) will be reported.\n";
|
|
5650 sleep (3);
|
|
5651 $directional = 0;
|
|
5652 }
|
|
5653 else{
|
|
5654 print "Library is assumed to be strand-specific (directional), alignments to strands complementary to the original top or bottom strands will be ignored (i.e. not performed!).\n";
|
|
5655 sleep (3);
|
|
5656 $directional = 1; # Changed this to being the default behaviour
|
|
5657 }
|
|
5658
|
|
5659 ### UNMAPPED SEQUENCE OUTPUT
|
|
5660 $unmapped = 0 unless ($unmapped);
|
|
5661
|
|
5662 ### AMBIGUOUS ALIGNMENT SEQUENCE OUTPUT
|
|
5663 $multi_map = 0 unless ($multi_map);
|
|
5664
|
|
5665
|
|
5666 ### OUTPUT DIRECTORY
|
|
5667
|
|
5668 chdir $parent_dir or die "Failed to move back to current working directory\n";
|
|
5669 if ($output_dir){
|
|
5670 unless ($output_dir =~ /\/$/){
|
|
5671 $output_dir =~ s/$/\//;
|
|
5672 }
|
|
5673
|
|
5674 if (chdir $output_dir){
|
|
5675 $output_dir = getcwd; # making the path absolute
|
|
5676 unless ($output_dir =~ /\/$/){
|
|
5677 $output_dir =~ s/$/\//;
|
|
5678 }
|
|
5679 }
|
|
5680 else{
|
|
5681 mkdir $output_dir or die "Unable to create directory $output_dir $!\n";
|
|
5682 warn "Created output directory $output_dir!\n\n";
|
|
5683 chdir $output_dir or die "Failed to move to $output_dir\n";
|
|
5684 $output_dir = getcwd; # making the path absolute
|
|
5685 unless ($output_dir =~ /\/$/){
|
|
5686 $output_dir =~ s/$/\//;
|
|
5687 }
|
|
5688 }
|
|
5689 warn "Output will be written into the directory: $output_dir\n";
|
|
5690 }
|
|
5691 else{
|
|
5692 $output_dir = '';
|
|
5693 }
|
|
5694
|
|
5695 ### TEMPORARY DIRECTORY for C->T and G->A transcribed files
|
|
5696
|
|
5697 chdir $parent_dir or die "Failed to move back to current working directory\n";
|
|
5698 if ($temp_dir){
|
|
5699 warn "\nUsing temp directory: $temp_dir\n";
|
|
5700 unless ($temp_dir =~ /\/$/){
|
|
5701 $temp_dir =~ s/$/\//;
|
|
5702 }
|
|
5703
|
|
5704 if (chdir $temp_dir){
|
|
5705 $temp_dir = getcwd; # making the path absolute
|
|
5706 unless ($temp_dir =~ /\/$/){
|
|
5707 $temp_dir =~ s/$/\//;
|
|
5708 }
|
|
5709 }
|
|
5710 else{
|
|
5711 mkdir $temp_dir or die "Unable to create directory $temp_dir $!\n";
|
|
5712 warn "Created temporary directory $temp_dir!\n\n";
|
|
5713 chdir $temp_dir or die "Failed to move to $temp_dir\n";
|
|
5714 $temp_dir = getcwd; # making the path absolute
|
|
5715 unless ($temp_dir =~ /\/$/){
|
|
5716 $temp_dir =~ s/$/\//;
|
|
5717 }
|
|
5718 }
|
|
5719 warn "Temporary files will be written into the directory: $temp_dir\n";
|
|
5720 }
|
|
5721 else{
|
|
5722 $temp_dir = '';
|
|
5723 }
|
|
5724
|
|
5725
|
|
5726 return ($genome_folder,$CT_index_basename,$GA_index_basename,$path_to_bowtie,$sequence_format,$bowtie_options,$directional,$unmapped,$multi_map,$phred64,$solexa,$output_dir,$bowtie2,$vanilla,$sam_no_hd,$skip,$qupto,$temp_dir);
|
|
5727 }
|
|
5728
|
|
5729
|
|
5730
|
|
5731 sub generate_SAM_header{
|
|
5732 print OUT "\@HD\tVN:1.0\tSO:unsorted\n"; # @HD = header, VN = version, SO = sort order
|
|
5733 foreach my $chr (keys %chromosomes){
|
|
5734 my $length = length ($chromosomes{$chr});
|
|
5735 print OUT "\@SQ\tSN:$chr\tLN:$length\n"; # @SQ = sequence, SN = seq name, LN = length
|
|
5736 }
|
|
5737 print OUT "\@PG\tID:Bismark\tVN:$bismark_version\tCL:\"bismark $command_line\"\n"; # @PG = program, ID = unique identifier, PN = program name name, VN = program version
|
|
5738 }
|
|
5739
|
|
5740 ### I would like to thank the following individuals for their valuable contributions to the Bismark SAM output format:
|
|
5741 ### O. Tam (Sep 2010), C. Whelan (2011), E. Vidal (2011), T. McBryan (2011), P. Hickey (2011)
|
|
5742
|
|
5743 sub single_end_SAM_output{
|
|
5744 my ($id,$actual_seq,$methylation_call_params,$qual) = @_;
|
|
5745 my $strand = $methylation_call_params->{$id}->{alignment_strand};
|
|
5746 my $chr = $methylation_call_params->{$id}->{chromosome};
|
|
5747 my $start = $methylation_call_params->{$id}->{position};
|
|
5748 my $stop = $methylation_call_params->{$id}->{end_position};
|
|
5749 my $ref_seq = $methylation_call_params->{$id}->{unmodified_genomic_sequence};
|
|
5750 my $methcall = $methylation_call_params->{$id}->{methylation_call};
|
|
5751 my $read_conversion = $methylation_call_params->{$id}->{read_conversion};
|
|
5752 my $genome_conversion = $methylation_call_params->{$id}->{genome_conversion};
|
|
5753 my $number_of_mismatches = $methylation_call_params->{$id}->{number_of_mismatches};
|
|
5754 ### This is a description of the bitwise FLAG field which needs to be set for the SAM file taken from: "The SAM Format Specification (v1.4-r985), September 7, 2011"
|
|
5755 ## FLAG: bitwise FLAG. Each bit is explained in the following table:
|
|
5756 ## Bit Description Comment Value
|
|
5757 ## 0x1 template has multiple segments in sequencing 0: single-end 1: paired end value: 2**0 ( 1)
|
|
5758 ## 0x2 each segment properly aligned according to the aligner true only for paired-end alignments value: 2**1 ( 2)
|
|
5759 ## 0x4 segment unmapped --- ---
|
|
5760 ## 0x8 next segment in the template unmapped --- ---
|
|
5761 ## 0x10 SEQ being reverse complemented value: 2**4 ( 16)
|
|
5762 ## 0x20 SEQ of the next segment in the template being reversed value: 2**5 ( 32)
|
|
5763 ## 0x40 the first segment in the template read 1 value: 2**6 ( 64)
|
|
5764 ## 0x80 the last segment in the template read 2 value: 2**7 (128)
|
|
5765 ## 0x100 secondary alignment --- ---
|
|
5766 ## 0x200 not passing quality controls --- ---
|
|
5767 ## 0x400 PCR or optical duplicate --- ---
|
|
5768
|
|
5769 #####
|
|
5770
|
|
5771 my $flag; # FLAG variable used for SAM format.
|
|
5772 if ($strand eq "+"){
|
|
5773 if ($read_conversion eq 'CT' and $genome_conversion eq 'CT'){
|
|
5774 $flag = 0; # 0 for "+" strand (OT)
|
|
5775 }
|
|
5776 elsif ($read_conversion eq 'GA' and $genome_conversion eq 'GA'){
|
|
5777 $flag = 16; # 16 for "-" strand (CTOB, yields information for the original bottom strand)
|
|
5778 }
|
|
5779 else{
|
|
5780 die "Unexpected strand and read/genome conversion: strand: $strand, read conversion: $read_conversion, genome_conversion: $genome_conversion\n\n";
|
|
5781 }
|
|
5782 }
|
|
5783 elsif ($strand eq "-"){
|
|
5784 if ($read_conversion eq 'CT' and $genome_conversion eq 'GA'){
|
|
5785 $flag = 16; # 16 for "-" strand (OB)
|
|
5786 }
|
|
5787 elsif ($read_conversion eq 'GA' and $genome_conversion eq 'CT'){
|
|
5788 $flag = 0; # 0 for "+" strand (CTOT, yields information for the original top strand)
|
|
5789 }
|
|
5790 else{
|
|
5791 die "Unexpected strand and read/genome conversion: strand: $strand, read conversion: $read_conversion, genome_conversion: $genome_conversion\n\n";
|
|
5792 }
|
|
5793 }
|
|
5794 else{
|
|
5795 die "Unexpected strand information: $strand\n\n";
|
|
5796 }
|
|
5797
|
|
5798 #####
|
|
5799
|
|
5800 my $mapq = 255; # Assume mapping quality is unavailable
|
|
5801
|
|
5802 #####
|
|
5803
|
|
5804 my $cigar;
|
|
5805 if ($bowtie2){
|
|
5806 $cigar = $methylation_call_params->{$id}->{CIGAR}; # Actual CIGAR string reported by Bowtie 2
|
|
5807 }
|
|
5808 else{
|
|
5809 $cigar = length($actual_seq) . "M"; # Bowtie 1 output does not contain indels (only matches and mismatches)
|
|
5810 }
|
|
5811
|
|
5812 #####
|
|
5813
|
|
5814 my $rnext = "*"; # Paired-end variable
|
|
5815
|
|
5816 #####
|
|
5817
|
|
5818 my $pnext = 0; # Paired-end variable
|
|
5819
|
|
5820 #####
|
|
5821
|
|
5822 my $tlen = 0; # Paired-end variable
|
|
5823
|
|
5824 #####
|
|
5825
|
|
5826 if ($read_conversion eq 'CT'){
|
|
5827 $ref_seq = substr($ref_seq, 0, length($ref_seq) - 2); # Removes additional nucleotides from the 3' end. This only works for the original top or bottom strands
|
|
5828 }
|
|
5829 else{
|
|
5830 $ref_seq = substr($ref_seq, 2, length($ref_seq) - 2); # Removes additional nucleotides from the 5' end. This works for the complementary strands in non-directional libraries
|
|
5831 }
|
|
5832
|
|
5833 if ($strand eq '-'){
|
|
5834 $actual_seq = revcomp($actual_seq); # Sequence represented on the forward genomic strand
|
|
5835 $ref_seq = revcomp($ref_seq); # Required for comparison with actual sequence
|
|
5836 $qual = reverse $qual; # if the sequence was reverse-complemented the quality string needs to be reversed as well
|
|
5837 }
|
|
5838
|
|
5839 #####
|
|
5840
|
|
5841 my $hemming_dist = hemming_dist($actual_seq,$ref_seq); # Edit distance to the reference, i.e. minimal number of one-nucleotide edits needed to transform the read string
|
|
5842 # into the reference string. hemming_dist()
|
|
5843 if ($bowtie2){
|
|
5844 $hemming_dist += $methylation_call_params->{$id}->{indels}; # Adding the number of inserted/deleted bases which we parsed while getting the genomic sequence
|
|
5845 }
|
|
5846
|
|
5847 my $NM_tag = "NM:i:$hemming_dist"; # Optional tag NM: edit distance based on nucleotide differences
|
|
5848
|
|
5849 #####
|
|
5850
|
|
5851 my $XX_tag = make_mismatch_string($actual_seq, $ref_seq); # Optional tag XX: string providing mismatched reference bases in the alignment (NO indel information!)
|
|
5852
|
|
5853 #####
|
|
5854
|
|
5855 my $XM_tag; # Optional tag XM: Methylation Call String
|
|
5856 if ($strand eq '+'){
|
|
5857 $XM_tag = "XM:Z:$methcall";
|
|
5858 }
|
|
5859 elsif ($strand eq '-'){
|
|
5860 $XM_tag = 'XM:Z:'.reverse $methcall; # if the sequence was reverse-complemented the methylation call string needs to be reversed as well
|
|
5861 }
|
|
5862
|
|
5863 #####
|
|
5864
|
|
5865 my $XR_tag = "XR:Z:$read_conversion"; # Optional tag XR: Read Conversion
|
|
5866
|
|
5867 #####
|
|
5868
|
|
5869 my $XG_tag = "XG:Z:$genome_conversion"; # Optional tag XG: Genome Conversion
|
|
5870
|
|
5871 #####
|
|
5872
|
|
5873 # SAM format: QNAME, FLAG, RNAME, 1-based POS, MAPQ, CIGAR, RNEXT, PNEXT, TLEN, SEQ, QUAL, optional fields
|
|
5874 print OUT join("\t",($id,$flag,$chr,$start,$mapq,$cigar,$rnext,$pnext,$tlen,$actual_seq,$qual,$NM_tag,$XX_tag,$XM_tag,$XR_tag,$XG_tag)),"\n";
|
|
5875 }
|
|
5876
|
|
5877
|
|
5878 sub paired_end_SAM_output{
|
|
5879 my ($id,$actual_seq_1,$actual_seq_2,$methylation_call_params,$qual_1,$qual_2) = @_;
|
|
5880 my $strand_1 = $methylation_call_params->{$id}->{alignment_read_1}; # Bowtie 1 only reports the read 1 alignment strand
|
|
5881 my $strand_2 = $methylation_call_params->{$id}->{alignment_read_2};
|
|
5882 my $chr = $methylation_call_params->{$id}->{chromosome};
|
|
5883 my $ref_seq_1 = $methylation_call_params->{$id}->{unmodified_genomic_sequence_1};
|
|
5884 my $ref_seq_2 = $methylation_call_params->{$id}->{unmodified_genomic_sequence_2};
|
|
5885 my $methcall_1 = $methylation_call_params->{$id}->{methylation_call_1};
|
|
5886 my $methcall_2 = $methylation_call_params->{$id}->{methylation_call_2};
|
|
5887 my $read_conversion_1 = $methylation_call_params->{$id}->{read_conversion_1};
|
|
5888 my $read_conversion_2 = $methylation_call_params->{$id}->{read_conversion_2};
|
|
5889 my $genome_conversion = $methylation_call_params->{$id}->{genome_conversion};
|
|
5890 my $number_of_mismatches_1 = $methylation_call_params->{$id}->{number_of_mismatches_1}; # only needed for custom allele-specific output, not the default!
|
|
5891 my $number_of_mismatches_2 = $methylation_call_params->{$id}->{number_of_mismatches_2};
|
|
5892
|
|
5893 my $id_1 = $id.'/1';
|
|
5894 my $id_2 = $id.'/2';
|
|
5895
|
|
5896 # Allows all degenerate nucleotide sequences in reference genome
|
|
5897 die "Reference sequence ($ref_seq_1) contains invalid nucleotides!\n" if $ref_seq_1 =~ /[^ACTGNRYMKSWBDHV]/i;
|
|
5898 die "Reference sequence ($ref_seq_2) contains invalid nucleotides!\n" if $ref_seq_2 =~ /[^ACTGNRYMKSWBDHV]/i;
|
|
5899
|
|
5900 my $index; # used to store the srand origin of the alignment in a less convoluted way
|
|
5901
|
|
5902 if ($read_conversion_1 eq 'CT' and $genome_conversion eq 'CT'){
|
|
5903 $index = 0; ## this is OT (original top strand)
|
|
5904 }
|
|
5905 elsif ($read_conversion_1 eq 'GA' and $genome_conversion eq 'GA'){
|
|
5906 $index = 1; ## this is CTOB (complementary to OB)
|
|
5907 }
|
|
5908 elsif ($read_conversion_1 eq 'GA' and $genome_conversion eq 'CT'){
|
|
5909 $index = 2; ## this is CTOT (complementary to OT)
|
|
5910 }
|
|
5911 elsif ($read_conversion_1 eq 'CT' and $genome_conversion eq 'GA'){
|
|
5912 $index = 3; ## this is OB (original bottom)
|
|
5913 }
|
|
5914 else {
|
|
5915 die "Unexpected combination of read 1 and genome conversion: $read_conversion_1 / $genome_conversion\n";
|
|
5916 }
|
|
5917
|
|
5918 ### we need to remove 2 bp of the genomic sequence as we were extracting read + 2bp long fragments to make a methylation call at the
|
|
5919 ### first or last position.
|
|
5920
|
|
5921 if ($index == 0 or $index == 3){ # OT or OB
|
|
5922 $ref_seq_1 = substr($ref_seq_1,0,length($ref_seq_1)-2);
|
|
5923 $ref_seq_2 = substr($ref_seq_2,2,length($ref_seq_2)-2);
|
|
5924 }
|
|
5925 else{ # CTOT or CTOB
|
|
5926 $ref_seq_1 = substr($ref_seq_1,2,length($ref_seq_1)-2);
|
|
5927 $ref_seq_2 = substr($ref_seq_2,0,length($ref_seq_2)-2);
|
|
5928 }
|
|
5929
|
|
5930 #####
|
|
5931
|
|
5932 my $start_read_1;
|
|
5933 my $start_read_2;
|
|
5934 # adjusting end positions
|
|
5935
|
|
5936 if ($bowtie2){
|
|
5937 $start_read_1 = $methylation_call_params->{$id}->{position_1};
|
|
5938 $start_read_2 = $methylation_call_params->{$id}->{position_2};
|
|
5939 }
|
|
5940 else{ # Bowtie 1 output. $strand_1 stores the alignment of Read 1
|
|
5941 if ($strand_1 eq '+'){ # Read 1 aligns to the + strand
|
|
5942 $start_read_1 = $methylation_call_params->{$id}->{start_seq_1};
|
|
5943 $start_read_2 = $methylation_call_params->{$id}->{alignment_end} - length ($actual_seq_2) + 1;
|
|
5944 }
|
|
5945 else{ # read 1 is on the - strand
|
|
5946 $start_read_1 = $methylation_call_params->{$id}->{alignment_end} - length ($actual_seq_1) + 1;
|
|
5947 $start_read_2 = $methylation_call_params->{$id}->{start_seq_1};
|
|
5948 }
|
|
5949 }
|
|
5950
|
|
5951 #####
|
|
5952
|
|
5953 my $end_read_1;
|
|
5954 my $end_read_2;
|
|
5955 # adjusting end positions
|
|
5956
|
|
5957 if ($bowtie2){
|
|
5958 $end_read_1 = $methylation_call_params->{$id}->{end_position_1};
|
|
5959 $end_read_2 = $methylation_call_params->{$id}->{end_position_2};
|
|
5960 }
|
|
5961 else{ # Bowtie 1 output. $strand_1 stores the alignment of Read 1
|
|
5962 if ($strand_1 eq '+'){ # Read 1 aligns to the + strand
|
|
5963 $end_read_1 = $methylation_call_params->{$id}->{start_seq_1} + length ($actual_seq_1)-1;
|
|
5964 $end_read_2 = $methylation_call_params->{$id}->{alignment_end};
|
|
5965 }
|
|
5966 else{
|
|
5967 $end_read_1 = $methylation_call_params->{$id}->{alignment_end};
|
|
5968 $end_read_2 = $methylation_call_params->{$id}->{start_seq_1} + length ($actual_seq_2)-1;
|
|
5969 }
|
|
5970 }
|
|
5971
|
|
5972 #####
|
|
5973
|
|
5974 ### This is a description of the bitwise FLAG field which needs to be set for the SAM file taken from: "The SAM Format Specification (v1.4-r985), September 7, 2011"
|
|
5975 ## FLAG: bitwise FLAG. Each bit is explained in the following table:
|
|
5976 ## Bit Description Comment Value
|
|
5977 ## 0x1 template having multiple segments in sequencing 0: single-end 1: paired end value: 2^^0 ( 1)
|
|
5978 ## 0x2 each segment properly aligned according to the aligner true only for paired-end alignments value: 2^^1 ( 2)
|
|
5979 ## 0x4 segment unmapped --- ---
|
|
5980 ## 0x8 next segment in the template unmapped --- ---
|
|
5981 ## 0x10 SEQ being reverse complemented - strand alignment value: 2^^4 ( 16)
|
|
5982 ## 0x20 SEQ of the next segment in the template being reversed + strand alignment value: 2^^5 ( 32)
|
|
5983 ## 0x40 the first segment in the template read 1 value: 2^^6 ( 64)
|
|
5984 ## 0x80 the last segment in the template read 2 value: 2^^7 (128)
|
|
5985 ## 0x100 secondary alignment --- ---
|
|
5986 ## 0x200 not passing quality controls --- ---
|
|
5987 ## 0x400 PCR or optical duplicate --- ---
|
|
5988
|
|
5989 ### As the FLAG value do not consider that there might be 4 different bisulfite strands of DNA, we are trying to make FLAG tags which take the strand identity into account
|
|
5990
|
|
5991 # strands OT and CTOT will be treated as aligning to the top strand (both sequences are scored as aligning to the top strand)
|
|
5992 # strands OB and CTOB will be treated as aligning to the bottom strand (both sequences are scored as reverse complemented sequences)
|
|
5993
|
|
5994 my $flag_1; # FLAG variable used for SAM format
|
|
5995 my $flag_2;
|
|
5996
|
|
5997 if ($index == 0){ # OT
|
|
5998 $flag_1 = 67; # Read 1 is on the + strand (1+2+64) (Read 2 is technically reverse-complemented, but we do not score it)
|
|
5999 $flag_2 = 131; # Read 2 is on - strand but informative for the OT (1+2+128)
|
|
6000 }
|
|
6001 elsif ($index == 1){ # CTOB
|
|
6002 $flag_1 = 115; # Read 1 is on the + strand, we score for OB (1+2+16+32+64)
|
|
6003 $flag_2 = 179; # Read 2 is on the - strand (1+2+16+32+128)
|
|
6004 }
|
|
6005 elsif ($index == 2){ # CTOT
|
|
6006 $flag_1 = 67; # Read 1 is on the - strand (CTOT) strand, but we score it for OT (1+2+64)
|
|
6007 $flag_2 = 131; # Read 2 is on the + strand, score it for OT (1+2+128)
|
|
6008 }
|
|
6009 elsif ($index == 3){ # OB
|
|
6010 $flag_1 = 115; # Read 1 is on the - strand, we score for OB (1+2+16+32+64)
|
|
6011 $flag_2 = 179; # Read 2 is on the + strand (1+2+16+32+128)
|
|
6012 }
|
|
6013
|
|
6014 #####
|
|
6015
|
|
6016 my $mapq = 255; # Mapping quality is unavailable
|
|
6017
|
|
6018 #####
|
|
6019
|
|
6020 my $cigar_1;
|
|
6021 my $cigar_2;
|
|
6022
|
|
6023 if ($bowtie2){
|
|
6024 $cigar_1 = $methylation_call_params->{$id}->{CIGAR_1}; # Actual CIGAR string reported by Bowtie 2
|
|
6025 $cigar_2 = $methylation_call_params->{$id}->{CIGAR_2};
|
|
6026 }
|
|
6027 else{
|
|
6028 $cigar_1 = length($actual_seq_1) . "M"; # Assume no indels for Bowtie 1 mapping (only matches and mismatches)
|
|
6029 $cigar_2 = length($actual_seq_2) . "M";
|
|
6030 }
|
|
6031
|
|
6032 #####
|
|
6033
|
|
6034 my $rnext = '='; # Chromosome of mate; applies to both reads
|
|
6035
|
|
6036 #####
|
|
6037
|
|
6038 my $pnext_1 = $start_read_2; # Leftmost position of mate
|
|
6039 my $pnext_2 = $start_read_1;
|
|
6040
|
|
6041 #####
|
|
6042
|
|
6043 my $tlen_1; # signed observed Template LENgth (or inferred fragment size)
|
|
6044 my $tlen_2;
|
|
6045
|
|
6046 if ($bowtie2){
|
|
6047
|
|
6048 if ($start_read_1 <= $start_read_2){
|
|
6049
|
|
6050 # Read 1 alignment is leftmost
|
|
6051
|
|
6052 if ($end_read_2 >= $end_read_1){
|
|
6053
|
|
6054 # -------------------------> read 1 reads overlapping
|
|
6055 # <------------------------- read 2
|
|
6056 #
|
|
6057 # or
|
|
6058 #
|
|
6059 # -------------------------> read 1
|
|
6060 # <----------------------- read 2 read 2 contained within read 1
|
|
6061 #
|
|
6062 # or
|
|
6063 #
|
|
6064 # -------------------------> read 1 reads 1 and 2 exactly overlapping
|
|
6065 # <------------------------- read 2
|
|
6066 #
|
|
6067
|
|
6068 # dovetailing of reads is not enabled for Bowtie 2 alignments
|
|
6069
|
|
6070 $tlen_1 = $end_read_2 - $start_read_1 + 1; # Leftmost read has a + sign,
|
|
6071 $tlen_2 = $start_read_1 - $end_read_2 - 1; # Rightmost read has a - sign
|
|
6072 }
|
|
6073 elsif ($end_read_2 < $end_read_1){
|
|
6074
|
|
6075 # -------------------------> read 1
|
|
6076 # <----------- read 2 read 2 contained within read 1
|
|
6077 #
|
|
6078 # or
|
|
6079 #
|
|
6080 # -------------------------> read 1
|
|
6081 # <----------- read 2 read 2 contained within read 1
|
|
6082
|
|
6083 # start and end of read 2 are fully contained within read 1
|
|
6084 $tlen_1 = 0; # Set as 0 when the information is unavailable
|
|
6085 $tlen_2 = 0; # Set as 0 when the information is unavailable
|
|
6086 }
|
|
6087
|
|
6088 }
|
|
6089
|
|
6090 elsif ($start_read_2 < $start_read_1){
|
|
6091
|
|
6092 if ($end_read_1 >= $end_read_2){
|
|
6093
|
|
6094 # Read 2 alignment is leftmost
|
|
6095
|
|
6096 # -------------------------> read 2 reads overlapping
|
|
6097 # <------------------------- read 1
|
|
6098 #
|
|
6099 # or
|
|
6100 #
|
|
6101 # -------------------------> read 2
|
|
6102 # <----------------------- read 1 read 1 contained within read 2
|
|
6103 #
|
|
6104 #
|
|
6105
|
|
6106 $tlen_2 = $end_read_1 - $start_read_2 + 1; # Leftmost read has a + sign,
|
|
6107 $tlen_1 = $start_read_2 - $end_read_1 - 1; # Rightmost read has a - sign
|
|
6108 }
|
|
6109 elsif ($end_read_1 < $end_read_2){
|
|
6110
|
|
6111 # -------------------------> read 2
|
|
6112 # <----------- read 1 read 1 contained within read 2
|
|
6113 #
|
|
6114 # or
|
|
6115 #
|
|
6116 # -------------------------> read 2
|
|
6117 # <----------- read 1 read 1 contained within read 2
|
|
6118
|
|
6119 # start and end of read 1 are fully contained within read 2
|
|
6120 $tlen_1 = 0; # Set as 0 when the information is unavailable
|
|
6121 $tlen_2 = 0; # Set as 0 when the information is unavailable
|
|
6122 }
|
|
6123 }
|
|
6124 }
|
|
6125
|
|
6126 else{ # Bowtie 1
|
|
6127
|
|
6128 if ($end_read_2 >= $end_read_1){
|
|
6129 # Read 1 alignment is leftmost
|
|
6130 # -------------------------> read 1
|
|
6131 # <------------------------- read 2
|
|
6132 # this is the most extreme case for Bowtie 1 alignments, reads do not contain each other, also no dovetailing
|
|
6133
|
|
6134 $tlen_1 = $end_read_2 - $start_read_1 + 1; # Leftmost read has a + sign,
|
|
6135 $tlen_2 = $start_read_1 - $end_read_2 - 1; # Rightmost read has a - sign
|
|
6136 }
|
|
6137 else{
|
|
6138 # Read 2 alignment is leftmost
|
|
6139 # -------------------------> read 2
|
|
6140 # <------------------------- read 1
|
|
6141 # this is the most extreme case for Bowtie 1 alignments, reads do not contain each other, also no dovetailing
|
|
6142
|
|
6143 $tlen_2 = $end_read_1 - $start_read_2 + 1; # Leftmost read has a + sign,
|
|
6144 $tlen_1 = $start_read_2 - $end_read_1 - 1; # Rightmost read has a - sign
|
|
6145 }
|
|
6146 }
|
|
6147
|
|
6148 #####
|
|
6149
|
|
6150 # adjusting the strand of the sequence before we use them to generate mismatch strings
|
|
6151 if ($strand_1 eq '-'){
|
|
6152 $actual_seq_1 = revcomp($actual_seq_1); # Sequence represented on the forward genomic strand
|
|
6153 $ref_seq_1 = revcomp($ref_seq_1); # Required for comparison with actual sequence
|
|
6154 $qual_1 = reverse $qual_1; # we need to reverse the quality string as well
|
|
6155 }
|
|
6156 if ($strand_2 eq '-'){
|
|
6157 $actual_seq_2 = revcomp($actual_seq_2); # Mate sequence represented on the forward genomic strand
|
|
6158 $ref_seq_2 = revcomp($ref_seq_2); # Required for comparison with actual sequence
|
|
6159 $qual_2 = reverse $qual_2; # If the sequence gets reverse complemented we reverse the quality string as well
|
|
6160 }
|
|
6161
|
|
6162 # print "$actual_seq_1\n$ref_seq_1\n\n";
|
|
6163 # print "$actual_seq_2\n$ref_seq_2\n\n";
|
|
6164
|
|
6165 #####
|
|
6166
|
|
6167 my $hemming_dist_1 = hemming_dist($actual_seq_1,$ref_seq_1); # Minimal number of one-nucleotide edits needed to transform the read string into the reference sequence
|
|
6168 my $hemming_dist_2 = hemming_dist($actual_seq_2,$ref_seq_2);
|
|
6169 if ($bowtie2){
|
|
6170 $hemming_dist_1 += $methylation_call_params->{$id}->{indels_1}; # Adding the number of inserted/deleted bases which we parsed while getting the genomic sequence
|
|
6171 $hemming_dist_2 += $methylation_call_params->{$id}->{indels_2}; # Adding the number of inserted/deleted bases which we parsed while getting the genomic sequence
|
|
6172 }
|
|
6173 my $NM_tag_1 = "NM:i:$hemming_dist_1"; # Optional tag NM: edit distance based on nucleotide differences
|
|
6174 my $NM_tag_2 = "NM:i:$hemming_dist_2"; # Optional tag NM: edit distance based on nucleotide differences
|
|
6175
|
|
6176 #####
|
|
6177
|
|
6178 my $XX_tag_1 = make_mismatch_string($actual_seq_1,$ref_seq_1); # Optional tag XX: String providing mismatched reference bases in the alignment (NO indel information!)
|
|
6179 my $XX_tag_2 = make_mismatch_string($actual_seq_2,$ref_seq_2);
|
|
6180
|
|
6181 #####
|
|
6182
|
|
6183 my $XM_tag_1; # Optional tag XM: Methylation call string
|
|
6184 my $XM_tag_2;
|
|
6185
|
|
6186 if ($strand_1 eq '-'){
|
|
6187 $XM_tag_1 = 'XM:Z:'.reverse $methcall_1; # Needs to be reversed if the sequence was reverse complemented
|
|
6188 }
|
|
6189 else{
|
|
6190 $XM_tag_1 = "XM:Z:$methcall_1";
|
|
6191 }
|
|
6192
|
|
6193 if ($strand_2 eq '-'){
|
|
6194 $XM_tag_2 = 'XM:Z:'.reverse $methcall_2; # Needs to be reversed if the sequence was reverse complemented
|
|
6195 }
|
|
6196 else{
|
|
6197 $XM_tag_2 = "XM:Z:$methcall_2";
|
|
6198 }
|
|
6199
|
|
6200 #####
|
|
6201
|
|
6202 my $XR_tag_1 = "XR:Z:$read_conversion_1"; # Optional tag XR: Read 1 conversion state
|
|
6203 my $XR_tag_2 = "XR:Z:$read_conversion_2"; # Optional tag XR: Read 2 conversion state
|
|
6204
|
|
6205 #####
|
|
6206
|
|
6207 my $XG_tag = "XG:Z:$genome_conversion"; # Optional tag XG: Genome Conversion state; valid for both reads
|
|
6208
|
|
6209 #####
|
|
6210
|
|
6211 # SAM format: QNAME, FLAG, RNAME, 1-based POS, MAPQ, CIGAR, RNEXT, PNEXT, TLEN, SEQ, QUAL, optional fields
|
|
6212 print OUT join("\t", ($id_1, $flag_1, $chr, $start_read_1, $mapq, $cigar_1, $rnext, $pnext_1, $tlen_1, $actual_seq_1, $qual_1, $NM_tag_1, $XX_tag_1, $XM_tag_1,$XR_tag_1,$XG_tag)), "\n";
|
|
6213 print OUT join("\t", ($id_2, $flag_2, $chr, $start_read_2, $mapq, $cigar_2, $rnext, $pnext_2, $tlen_2, $actual_seq_2, $qual_2, $NM_tag_2, $XX_tag_2, $XM_tag_2,$XR_tag_2,$XG_tag)), "\n";
|
|
6214 }
|
|
6215
|
|
6216 sub revcomp{
|
|
6217 my $seq = shift or die "Missing seq to reverse complement\n";
|
|
6218 $seq = reverse $seq;
|
|
6219 $seq =~ tr/ACTGactg/TGACTGAC/;
|
|
6220 return $seq;
|
|
6221 }
|
|
6222
|
|
6223 sub hemming_dist{
|
|
6224 my $matches = 0;
|
|
6225 my @actual_seq = split //,(shift @_);
|
|
6226 my @ref_seq = split //,(shift @_);
|
|
6227 foreach (0..$#actual_seq){
|
|
6228 ++$matches if ($actual_seq[$_] eq $ref_seq[$_]);
|
|
6229 }
|
|
6230 return my $hd = scalar @actual_seq - $matches;
|
|
6231 }
|
|
6232
|
|
6233 sub make_mismatch_string{
|
|
6234 my $actual_seq = shift or die "Missing actual sequence";
|
|
6235 my $ref_seq = shift or die "Missing reference sequence";
|
|
6236 my $XX_tag = "XX:Z:";
|
|
6237 my $tmp = ($actual_seq ^ $ref_seq); # Bitwise comparison
|
|
6238 my $prev_mm_pos = 0;
|
|
6239 while($tmp =~ /[^\0]/g){ # Where bitwise comparison showed a difference
|
|
6240 my $nuc_match = pos($tmp) - $prev_mm_pos - 1; # Generate number of nucleotide that matches since last mismatch
|
|
6241 my $nuc_mm = substr($ref_seq, pos($tmp) - 1, 1) if pos($tmp) <= length($ref_seq); # Obtain reference nucleotide that was different from the actual read
|
|
6242 $XX_tag .= "$nuc_match" if $nuc_match > 0; # Ignore if mismatches are adjacent to each other
|
|
6243 $XX_tag .= "$nuc_mm" if defined $nuc_mm; # Ignore if there is no mismatch (prevents uninitialized string concatenation)
|
|
6244 $prev_mm_pos = pos($tmp); # Position of last mismatch
|
|
6245 }
|
|
6246 my $end_matches = length($ref_seq) - $prev_mm_pos; # Provides number of matches from last mismatch till end of sequence
|
|
6247 $XX_tag .= "$end_matches" if $end_matches > 0; # Ignore if mismatch is at the end of sequence
|
|
6248 return $XX_tag;
|
|
6249 }
|
|
6250
|
|
6251
|
|
6252
|
|
6253 sub print_helpfile{
|
|
6254 print << "HOW_TO";
|
|
6255
|
|
6256
|
|
6257 This program is free software: you can redistribute it and/or modify
|
|
6258 it under the terms of the GNU General Public License as published by
|
|
6259 the Free Software Foundation, either version 3 of the License, or
|
|
6260 (at your option) any later version.
|
|
6261
|
|
6262 This program is distributed in the hope that it will be useful,
|
|
6263 but WITHOUT ANY WARRANTY; without even the implied warranty of
|
|
6264 MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the
|
|
6265 GNU General Public License for more details.
|
|
6266 You should have received a copy of the GNU General Public License
|
|
6267 along with this program. If not, see <http://www.gnu.org/licenses/>.
|
|
6268
|
|
6269
|
|
6270
|
|
6271 DESCRIPTION
|
|
6272
|
|
6273
|
|
6274 The following is a brief description of command line options and arguments to control the Bismark
|
|
6275 bisulfite mapper and methylation caller. Bismark takes in FastA or FastQ files and aligns the
|
|
6276 reads to a specified bisulfite genome. Sequence reads are transformed into a bisulfite converted forward strand
|
|
6277 version (C->T conversion) or into a bisulfite treated reverse strand (G->A conversion of the forward strand).
|
|
6278 Each of these reads are then aligned to bisulfite treated forward strand index of a reference genome
|
|
6279 (C->T converted) and a bisulfite treated reverse strand index of the genome (G->A conversion of the
|
|
6280 forward strand, by doing this alignments will produce the same positions). These 4 instances of Bowtie (1 or 2)
|
|
6281 are run in parallel. The sequence file(s) are then read in again sequence by sequence to pull out the original
|
|
6282 sequence from the genome and determine if there were any protected C's present or not.
|
|
6283
|
|
6284 As of version 0.7.0 Bismark will only run 2 alignment threads for OT and OB in parallel, the 4 strand mode can be
|
|
6285 re-enabled by using --non_directional.
|
|
6286
|
|
6287 The final output of Bismark is in SAM format by default. For Bowtie 1 one can alos choose to report the old
|
|
6288 'vanilla' output format, which is a single tab delimited file with all sequences that have a unique best
|
|
6289 alignment to any of the 4 possible strands of a bisulfite PCR product. Both formats are described in more detail below.
|
|
6290
|
|
6291
|
|
6292 USAGE: bismark [options] <genome_folder> {-1 <mates1> -2 <mates2> | <singles>}
|
|
6293
|
|
6294
|
|
6295 ARGUMENTS:
|
|
6296
|
|
6297 <genome_folder> The path to the folder containing the unmodified reference genome
|
|
6298 as well as the subfolders created by the Bismark_Genome_Preparation
|
|
6299 script (/Bisulfite_Genome/CT_conversion/ and /Bisulfite_Genome/GA_conversion/).
|
|
6300 Bismark expects one or more fastA files in this folder (file extension: .fa
|
|
6301 or .fasta). The path can be relative or absolute.
|
|
6302
|
|
6303 -1 <mates1> Comma-separated list of files containing the #1 mates (filename usually includes
|
|
6304 "_1"), e.g. flyA_1.fq,flyB_1.fq). Sequences specified with this option must
|
|
6305 correspond file-for-file and read-for-read with those specified in <mates2>.
|
|
6306 Reads may be a mix of different lengths. Bismark will produce one mapping result
|
|
6307 and one report file per paired-end input file pair.
|
|
6308
|
|
6309 -2 <mates2> Comma-separated list of files containing the #2 mates (filename usually includes
|
|
6310 "_2"), e.g. flyA_1.fq,flyB_1.fq). Sequences specified with this option must
|
|
6311 correspond file-for-file and read-for-read with those specified in <mates1>.
|
|
6312 Reads may be a mix of different lengths.
|
|
6313
|
|
6314 <singles> A comma- or space-separated list of files containing the reads to be aligned (e.g.
|
|
6315 lane1.fq,lane2.fq lane3.fq). Reads may be a mix of different lengths. Bismark will
|
|
6316 produce one mapping result and one report file per input file.
|
|
6317
|
|
6318
|
|
6319 OPTIONS:
|
|
6320
|
|
6321
|
|
6322 Input:
|
|
6323
|
|
6324 -q/--fastq The query input files (specified as <mate1>,<mate2> or <singles> are FASTQ
|
|
6325 files (usually having extension .fg or .fastq). This is the default. See also
|
|
6326 --solexa-quals.
|
|
6327
|
|
6328 -f/--fasta The query input files (specified as <mate1>,<mate2> or <singles> are FASTA
|
|
6329 files (usually havin extension .fa, .mfa, .fna or similar). All quality values
|
|
6330 are assumed to be 40 on the Phred scale.
|
|
6331
|
|
6332 -s/--skip <int> Skip (i.e. do not align) the first <int> reads or read pairs from the input.
|
|
6333
|
|
6334 -u/--upto <int> Only aligns the first <int> reads or read pairs from the input. Default: no limit.
|
|
6335
|
|
6336 --phred33-quals FASTQ qualities are ASCII chars equal to the Phred quality plus 33. Default: on.
|
|
6337
|
|
6338 --phred64-quals FASTQ qualities are ASCII chars equal to the Phred quality plus 64. Default: off.
|
|
6339
|
|
6340 --solexa-quals Convert FASTQ qualities from solexa-scaled (which can be negative) to phred-scaled
|
|
6341 (which can't). The formula for conversion is:
|
|
6342 phred-qual = 10 * log(1 + 10 ** (solexa-qual/10.0)) / log(10). Used with -q. This
|
|
6343 is usually the right option for use with (unconverted) reads emitted by the GA
|
|
6344 Pipeline versions prior to 1.3. Works only for Bowtie 1. Default: off.
|
|
6345
|
|
6346 --solexa1.3-quals Same as --phred64-quals. This is usually the right option for use with (unconverted)
|
|
6347 reads emitted by GA Pipeline version 1.3 or later. Default: off.
|
|
6348
|
|
6349 --path_to_bowtie The full path </../../> to the Bowtie (1 or 2) installation on your system. If not
|
|
6350 specified it is assumed that Bowtie (1 or 2) is in the PATH.
|
|
6351
|
|
6352
|
|
6353 Alignment:
|
|
6354
|
|
6355 -n/--seedmms <int> The maximum number of mismatches permitted in the "seed", i.e. the first L base pairs
|
|
6356 of the read (where L is set with -l/--seedlen). This may be 0, 1, 2 or 3 and the
|
|
6357 default is 1. This option is only available for Bowtie 1 (for Bowtie 2 see -N).
|
|
6358
|
|
6359 -l/--seedlen The "seed length"; i.e., the number of bases of the high quality end of the read to
|
|
6360 which the -n ceiling applies. The default is 28. Bowtie (and thus Bismark) is faster for
|
|
6361 larger values of -l. This option is only available for Bowtie 1 (for Bowtie 2 see -L).
|
|
6362
|
|
6363 -e/--maqerr <int> Maximum permitted total of quality values at all mismatched read positions throughout
|
|
6364 the entire alignment, not just in the "seed". The default is 70. Like Maq, bowtie rounds
|
|
6365 quality values to the nearest 10 and saturates at 30. This value is not relevant for
|
|
6366 Bowtie 2.
|
|
6367
|
|
6368 --chunkmbs <int> The number of megabytes of memory a given thread is given to store path descriptors in
|
|
6369 --best mode. Best-first search must keep track of many paths at once to ensure it is
|
|
6370 always extending the path with the lowest cumulative cost. Bowtie tries to minimize the
|
|
6371 memory impact of the descriptors, but they can still grow very large in some cases. If
|
|
6372 you receive an error message saying that chunk memory has been exhausted in --best mode,
|
|
6373 try adjusting this parameter up to dedicate more memory to the descriptors. This value
|
|
6374 is not relevant for Bowtie 2. Default: 512.
|
|
6375
|
|
6376 -I/--minins <int> The minimum insert size for valid paired-end alignments. E.g. if -I 60 is specified and
|
|
6377 a paired-end alignment consists of two 20-bp alignments in the appropriate orientation
|
|
6378 with a 20-bp gap between them, that alignment is considered valid (as long as -X is also
|
|
6379 satisfied). A 19-bp gap would not be valid in that case. Default: 0.
|
|
6380
|
|
6381 -X/--maxins <int> The maximum insert size for valid paired-end alignments. E.g. if -X 100 is specified and
|
|
6382 a paired-end alignment consists of two 20-bp alignments in the proper orientation with a
|
|
6383 60-bp gap between them, that alignment is considered valid (as long as -I is also satisfied).
|
|
6384 A 61-bp gap would not be valid in that case. Default: 500.
|
|
6385
|
|
6386
|
|
6387 Bowtie 1 Reporting:
|
|
6388
|
|
6389 -k <2> Due to the way Bismark works Bowtie will report up to 2 valid alignments. This option
|
|
6390 will be used by default.
|
|
6391
|
|
6392 --best Make Bowtie guarantee that reported singleton alignments are "best" in terms of stratum
|
|
6393 (i.e. number of mismatches, or mismatches in the seed in the case if -n mode) and in
|
|
6394 terms of the quality; e.g. a 1-mismatch alignment where the mismatch position has Phred
|
|
6395 quality 40 is preferred over a 2-mismatch alignment where the mismatched positions both
|
|
6396 have Phred quality 10. When --best is not specified, Bowtie may report alignments that
|
|
6397 are sub-optimal in terms of stratum and/or quality (though an effort is made to report
|
|
6398 the best alignment). --best mode also removes all strand bias. Note that --best does not
|
|
6399 affect which alignments are considered "valid" by Bowtie, only which valid alignments
|
|
6400 are reported by Bowtie. Bowtie is about 1-2.5 times slower when --best is specified.
|
|
6401 Default: on.
|
|
6402
|
|
6403 --no_best Disables the --best option which is on by default. This can speed up the alignment process,
|
|
6404 e.g. for testing purposes, but for credible results it is not recommended to disable --best.
|
|
6405
|
|
6406
|
|
6407 Output:
|
|
6408
|
|
6409 --non_directional The sequencing library was constructed in a non strand-specific manner, alignments to all four
|
|
6410 bisulfite strands will be reported. Default: OFF.
|
|
6411
|
|
6412 (The current Illumina protocol for BS-Seq is directional, in which case the strands complementary
|
|
6413 to the original strands are merely theoretical and should not exist in reality. Specifying directional
|
|
6414 alignments (which is the default) will only run 2 alignment threads to the original top (OT)
|
|
6415 or bottom (OB) strands in parallel and report these alignments. This is the recommended option
|
|
6416 for sprand-specific libraries).
|
|
6417
|
|
6418 --sam-no-hd Suppress SAM header lines (starting with @). This might be useful when very large input files are
|
|
6419 split up into several smaller files to run concurrently and the output files are to be merged.
|
|
6420
|
|
6421 --quiet Print nothing besides alignments.
|
|
6422
|
|
6423 --vanilla Performs bisulfite mapping with Bowtie 1 and prints the 'old' output (as in Bismark 0.5.X) instead
|
|
6424 of SAM format output.
|
|
6425
|
|
6426 -un/--unmapped Write all reads that could not be aligned to a file in the output directory. Written reads will
|
|
6427 appear as they did in the input, without any translation of quality values that may have
|
|
6428 taken place within Bowtie or Bismark. Paired-end reads will be written to two parallel files with _1
|
|
6429 and _2 inserted in their filenames, i.e. _unmapped_reads_1.txt and unmapped_reads_2.txt. Reads
|
|
6430 with more than one valid alignment with the same number of lowest mismatches (ambiguous mapping)
|
|
6431 are also written to _unmapped_reads.txt unless the option --ambiguous is specified as well.
|
|
6432
|
|
6433 --ambiguous Write all reads which produce more than one valid alignment with the same number of lowest
|
|
6434 mismatches or other reads that fail to align uniquely to a file in the output directory.
|
|
6435 Written reads will appear as they did in the input, without any of the translation of quality
|
|
6436 values that may have taken place within Bowtie or Bismark. Paired-end reads will be written to two
|
|
6437 parallel files with _1 and _2 inserted in theit filenames, i.e. _ambiguous_reads_1.txt and
|
|
6438 _ambiguous_reads_2.txt. These reads are not written to the file specified with --un.
|
|
6439
|
|
6440 -o/--output_dir <dir> Write all output files into this directory. By default the output files will be written into
|
|
6441 the same folder as the input file(s). If the specified folder does not exist, Bismark will attempt
|
|
6442 to create it first. The path to the output folder can be either relative or absolute.
|
|
6443
|
|
6444 --temp_dir <dir> Write temporary files to this directory instead of into the same directory as the input files. If
|
|
6445 the specified folder does not exist, Bismark will attempt to create it first. The path to the
|
|
6446 temporary folder can be either relative or absolute.
|
|
6447
|
|
6448
|
|
6449
|
|
6450 Other:
|
|
6451
|
|
6452 -h/--help Displays this help file.
|
|
6453
|
|
6454 -v/--version Displays version information.
|
|
6455
|
|
6456
|
|
6457 BOWTIE 2 SPECIFIC OPTIONS
|
|
6458
|
|
6459 --bowtie2 Uses Bowtie 2 instead of Bowtie 1. Bismark limits Bowtie 2 to only perform end-to-end
|
|
6460 alignments, i.e. searches for alignments involving all read characters (also called
|
|
6461 untrimmed or unclipped alignments). Bismark assumes that raw sequence data is adapter
|
|
6462 and/or quality trimmed where appropriate. Default: off.
|
|
6463
|
|
6464 Bowtie 2 alignment options:
|
|
6465
|
|
6466 -N <int> Sets the number of mismatches to allowed in a seed alignment during multiseed alignment.
|
|
6467 Can be set to 0 or 1. Setting this higher makes alignment slower (often much slower)
|
|
6468 but increases sensitivity. Default: 0. This option is only available for Bowtie 2 (for
|
|
6469 Bowtie 1 see -n).
|
|
6470
|
|
6471 -L <int> Sets the length of the seed substrings to align during multiseed alignment. Smaller values
|
|
6472 make alignment slower but more senstive. Default: the --sensitive preset of Bowtie 2 is
|
|
6473 used by default, which sets -L to 20. This option is only available for Bowtie 2 (for
|
|
6474 Bowtie 1 see -l).
|
|
6475
|
|
6476 --ignore-quals When calculating a mismatch penalty, always consider the quality value at the mismatched
|
|
6477 position to be the highest possible, regardless of the actual value. I.e. input is treated
|
|
6478 as though all quality values are high. This is also the default behavior when the input
|
|
6479 doesn't specify quality values (e.g. in -f mode). This option is invariable and on by default.
|
|
6480
|
|
6481
|
|
6482 Bowtie 2 paired-end options:
|
|
6483
|
|
6484 --no-mixed This option disables Bowtie 2's behavior to try to find alignments for the individual mates if
|
|
6485 it cannot find a concordant or discordant alignment for a pair. This option is invariable and
|
|
6486 and on by default.
|
|
6487
|
|
6488 --no-discordant Normally, Bowtie 2 looks for discordant alignments if it cannot find any concordant alignments.
|
|
6489 A discordant alignment is an alignment where both mates align uniquely, but that does not
|
|
6490 satisfy the paired-end constraints (--fr/--rf/--ff, -I, -X). This option disables that behavior
|
|
6491 and it is on by default.
|
|
6492
|
|
6493
|
|
6494 Bowtie 2 effort options:
|
|
6495
|
|
6496 -D <int> Up to <int> consecutive seed extension attempts can "fail" before Bowtie 2 moves on, using
|
|
6497 the alignments found so far. A seed extension "fails" if it does not yield a new best or a
|
|
6498 new second-best alignment. Default: 15.
|
|
6499
|
|
6500 -R <int> <int> is the maximum number of times Bowtie 2 will "re-seed" reads with repetitive seeds.
|
|
6501 When "re-seeding," Bowtie 2 simply chooses a new set of reads (same length, same number of
|
|
6502 mismatches allowed) at different offsets and searches for more alignments. A read is considered
|
|
6503 to have repetitive seeds if the total number of seed hits divided by the number of seeds
|
|
6504 that aligned at least once is greater than 300. Default: 2.
|
|
6505
|
|
6506 Bowtie 2 parallelization options:
|
|
6507
|
|
6508
|
|
6509 -p NTHREADS Launch NTHREADS parallel search threads (default: 1). Threads will run on separate processors/cores
|
|
6510 and synchronize when parsing reads and outputting alignments. Searching for alignments is highly
|
|
6511 parallel, and speedup is close to linear. Increasing -p increases Bowtie 2's memory footprint.
|
|
6512 E.g. when aligning to a human genome index, increasing -p from 1 to 8 increases the memory footprint
|
|
6513 by a few hundred megabytes. This option is only available if bowtie is linked with the pthreads
|
|
6514 library (i.e. if BOWTIE_PTHREADS=0 is not specified at build time). In addition, this option will
|
|
6515 automatically use the option '--reorder', which guarantees that output SAM records are printed in
|
|
6516 an order corresponding to the order of the reads in the original input file, even when -p is set
|
|
6517 greater than 1 (Bismark requires the Bowtie 2 output to be this way). Specifying --reorder and
|
|
6518 setting -p greater than 1 causes Bowtie 2 to run somewhat slower and use somewhat more memory then
|
|
6519 if --reorder were not specified. Has no effect if -p is set to 1, since output order will naturally
|
|
6520 correspond to input order in that case.
|
|
6521
|
|
6522 Bowtie 2 Scoring options:
|
|
6523
|
|
6524 --score_min <func> Sets a function governing the minimum alignment score needed for an alignment to be considered
|
|
6525 "valid" (i.e. good enough to report). This is a function of read length. For instance, specifying
|
|
6526 L,0,-0.2 sets the minimum-score function f to f(x) = 0 + -0.2 * x, where x is the read length.
|
|
6527 See also: setting function options at http://bowtie-bio.sourceforge.net/bowtie2. The default is
|
|
6528 L,0,-0.2.
|
|
6529
|
|
6530 --rdg <int1>,<int2> Sets the read gap open (<int1>) and extend (<int2>) penalties. A read gap of length N gets a penalty
|
|
6531 of <int1> + N * <int2>. Default: 5, 3.
|
|
6532
|
|
6533 --rfg <int1>,<int2> Sets the reference gap open (<int1>) and extend (<int2>) penalties. A reference gap of length N gets
|
|
6534 a penalty of <int1> + N * <int2>. Default: 5, 3.
|
|
6535
|
|
6536
|
|
6537 Bowtie 2 Reporting options:
|
|
6538
|
|
6539 -most_valid_alignments <int> This used to be the Bowtie 2 parameter -M. As of Bowtie 2 version 2.0.0 beta7 the option -M is
|
|
6540 deprecated. It will be removed in subsequent versions. What used to be called -M mode is still the
|
|
6541 default mode, but adjusting the -M setting is deprecated. Use the -D and -R options to adjust the
|
|
6542 effort expended to find valid alignments.
|
|
6543
|
|
6544 For reference, this used to be the old (now deprecated) description of -M:
|
|
6545 Bowtie 2 searches for at most <int>+1 distinct, valid alignments for each read. The search terminates when it
|
|
6546 can't find more distinct valid alignments, or when it finds <int>+1 distinct alignments, whichever
|
|
6547 happens first. Only the best alignment is reported. Information from the other alignments is used to
|
|
6548 estimate mapping quality and to set SAM optional fields, such as AS:i and XS:i. Increasing -M makes
|
|
6549 Bowtie 2 slower, but increases the likelihood that it will pick the correct alignment for a read that
|
|
6550 aligns many places. For reads that have more than <int>+1 distinct, valid alignments, Bowtie 2 does not
|
|
6551 guarantee that the alignment reported is the best possible in terms of alignment score. -M is
|
|
6552 always used and its default value is set to 10.
|
|
6553
|
|
6554
|
|
6555 'VANILLA' Bismark OUTPUT:
|
|
6556
|
|
6557 Single-end output format (tab-separated):
|
|
6558
|
|
6559 (1) <seq-ID>
|
|
6560 (2) <read alignment strand>
|
|
6561 (3) <chromosome>
|
|
6562 (4) <start position>
|
|
6563 (5) <end position>
|
|
6564 (6) <observed bisulfite sequence>
|
|
6565 (7) <equivalent genomic sequence>
|
|
6566 (8) <methylation call>
|
|
6567 (9) <read conversion
|
|
6568 (10) <genome conversion>
|
|
6569 (11) <read quality score (Phred33)>
|
|
6570
|
|
6571
|
|
6572 Paired-end output format (tab-separated):
|
|
6573 (1) <seq-ID>
|
|
6574 (2) <read 1 alignment strand>
|
|
6575 (3) <chromosome>
|
|
6576 (4) <start position>
|
|
6577 (5) <end position>
|
|
6578 (6) <observed bisulfite sequence 1>
|
|
6579 (7) <equivalent genomic sequence 1>
|
|
6580 (8) <methylation call 1>
|
|
6581 (9) <observed bisulfite sequence 2>
|
|
6582 (10) <equivalent genomic sequence 2>
|
|
6583 (11) <methylation call 2>
|
|
6584 (12) <read 1 conversion
|
|
6585 (13) <genome conversion>
|
|
6586 (14) <read 1 quality score (Phred33)>
|
|
6587 (15) <read 2 quality score (Phred33)>
|
|
6588
|
|
6589
|
|
6590 Bismark SAM OUTPUT (default):
|
|
6591
|
|
6592 (1) QNAME (seq-ID)
|
|
6593 (2) FLAG (this flag tries to take the strand a bisulfite read originated from into account (this is different from ordinary DNA alignment flags!))
|
|
6594 (3) RNAME (chromosome)
|
|
6595 (4) POS (start position)
|
|
6596 (5) MAPQ (always 255)
|
|
6597 (6) CIGAR
|
|
6598 (7) RNEXT
|
|
6599 (8) PNEXT
|
|
6600 (9) TLEN
|
|
6601 (10) SEQ
|
|
6602 (11) QUAL (Phred33 scale)
|
|
6603 (12) NM-tag (edit distance to the reference)
|
|
6604 (13) XX-tag (base-by-base mismatches to the reference. This does not include indels)
|
|
6605 (14) XM-tag (methylation call string)
|
|
6606 (15) XR-tag (read conversion state for the alignment)
|
|
6607 (16) XG-tag (genome conversion state for the alignment)
|
|
6608
|
|
6609 Each read of paired-end alignments is written out in a separate line in the above format.
|
|
6610
|
|
6611
|
|
6612 This script was last edited on 21 Aug 2012.
|
|
6613
|
|
6614 HOW_TO
|
|
6615 }
|