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10
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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@bbsrc.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 = 'v0.7.6';
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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 $amb_2\n";
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358 }
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359
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|
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360 if ($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
|
|
|
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 $sam_no_hd){
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372 generate_SAM_header();
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|
|
373 }
|
|
|
374
|
|
|
375 ### Input files are in FastA format
|
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|
376 if ($sequence_file_format eq 'FASTA'){
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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);
|
|
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378 }
|
|
|
379 ### Input files are in FastQ format
|
|
|
380 else{
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|
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
|
|
|
423 my $percent_alignable_sequences = sprintf ("%.1f",$counting{unique_best_alignment_count}*100/$counting{sequences_count});
|
|
|
424
|
|
|
425 warn "Number of alignments with a unique best hit from the different alignments:\t$counting{unique_best_alignment_count}\nMapping efficiency:\t${percent_alignable_sequences}%\n\n";
|
|
|
426 print REPORT "Number of alignments with a unique best hit from the different alignments:\t$counting{unique_best_alignment_count}\nMapping efficiency:\t${percent_alignable_sequences}%\n";
|
|
|
427
|
|
|
428 ### percentage of low complexity reads overruled because of low complexity (thereby creating a bias for highly methylated reads),
|
|
|
429 ### only calculating the percentage if there were any overruled alignments
|
|
|
430 if ($counting{low_complexity_alignments_overruled_count}){
|
|
|
431 my $percent_overruled_low_complexity_alignments = sprintf ("%.1f",$counting{low_complexity_alignments_overruled_count}*100/$counting{sequences_count});
|
|
|
432 # print REPORT "Number of low complexity alignments which were overruled to have a unique best hit rather than discarding them:\t$counting{low_complexity_alignments_overruled_count}\t(${percent_overruled_low_complexity_alignments}%)\n";
|
|
|
433 }
|
|
|
434
|
|
|
435 print "Sequences with no alignments under any condition:\t$counting{no_single_alignment_found}\n";
|
|
|
436 print "Sequences did not map uniquely:\t$counting{unsuitable_sequence_count}\n";
|
|
|
437 print "Sequences which were discarded because genomic sequence could not be extracted:\t$counting{genomic_sequence_could_not_be_extracted_count}\n\n";
|
|
|
438 print "Number of sequences with unique best (first) alignment came from the bowtie output:\n";
|
|
|
439 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";
|
|
|
440
|
|
|
441 print REPORT "Sequences with no alignments under any condition:\t$counting{no_single_alignment_found}\n";
|
|
|
442 print REPORT "Sequences did not map uniquely:\t$counting{unsuitable_sequence_count}\n";
|
|
|
443 print REPORT "Sequences which were discarded because genomic sequence could not be extracted:\t$counting{genomic_sequence_could_not_be_extracted_count}\n\n";
|
|
|
444 print REPORT "Number of sequences with unique best (first) alignment came from the bowtie output:\n";
|
|
|
445 print REPORT join ("\n","CT/CT:\t$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$counting{GA_GA_count}\t(complementary to (converted) bottom strand)"),"\n\n";
|
|
|
446
|
|
|
447 if ($directional){
|
|
|
448 print "Number of alignments to (merely theoretical) complementary strands being rejected in total:\t$counting{alignments_rejected_count}\n\n";
|
|
|
449 print REPORT "Number of alignments to (merely theoretical) complementary strands being rejected in total:\t$counting{alignments_rejected_count}\n\n";
|
|
|
450 }
|
|
|
451
|
|
|
452 ### detailed information about Cs analysed
|
|
|
453 warn "Final Cytosine Methylation Report\n",'='x33,"\n";
|
|
|
454 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};
|
|
|
455 warn "Total number of C's analysed:\t$total_number_of_C\n\n";
|
|
|
456 warn "Total methylated C's in CpG context:\t$counting{total_meCpG_count}\n";
|
|
|
457 warn "Total methylated C's in CHG context:\t$counting{total_meCHG_count}\n";
|
|
|
458 warn "Total methylated C's in CHH context:\t$counting{total_meCHH_count}\n\n";
|
|
|
459 warn "Total C to T conversions in CpG context:\t$counting{total_unmethylated_CpG_count}\n";
|
|
|
460 warn "Total C to T conversions in CHG context:\t$counting{total_unmethylated_CHG_count}\n";
|
|
|
461 warn "Total C to T conversions in CHH context:\t$counting{total_unmethylated_CHH_count}\n\n";
|
|
|
462
|
|
|
463 print REPORT "Final Cytosine Methylation Report\n",'='x33,"\n";
|
|
|
464 print REPORT "Total number of C's analysed:\t$total_number_of_C\n\n";
|
|
|
465 print REPORT "Total methylated C's in CpG context:\t $counting{total_meCpG_count}\n";
|
|
|
466 print REPORT "Total methylated C's in CHG context:\t$counting{total_meCHG_count}\n";
|
|
|
467 print REPORT "Total methylated C's in CHH context:\t$counting{total_meCHH_count}\n\n";
|
|
|
468 print REPORT "Total C to T conversions in CpG context:\t$counting{total_unmethylated_CpG_count}\n";
|
|
|
469 print REPORT "Total C to T conversions in CHG context:\t$counting{total_unmethylated_CHG_count}\n";
|
|
|
470 print REPORT "Total C to T conversions in CHH context:\t$counting{total_unmethylated_CHH_count}\n\n";
|
|
|
471
|
|
|
472 my $percent_meCHG;
|
|
|
473 if (($counting{total_meCHG_count}+$counting{total_unmethylated_CHG_count}) > 0){
|
|
|
474 $percent_meCHG = sprintf("%.1f",100*$counting{total_meCHG_count}/($counting{total_meCHG_count}+$counting{total_unmethylated_CHG_count}));
|
|
|
475 }
|
|
|
476
|
|
|
477 my $percent_meCHH;
|
|
|
478 if (($counting{total_meCHH_count}+$counting{total_unmethylated_CHH_count}) > 0){
|
|
|
479 $percent_meCHH = sprintf("%.1f",100*$counting{total_meCHH_count}/($counting{total_meCHH_count}+$counting{total_unmethylated_CHH_count}));
|
|
|
480 }
|
|
|
481
|
|
|
482 my $percent_meCpG;
|
|
|
483 if (($counting{total_meCpG_count}+$counting{total_unmethylated_CpG_count}) > 0){
|
|
|
484 $percent_meCpG = sprintf("%.1f",100*$counting{total_meCpG_count}/($counting{total_meCpG_count}+$counting{total_unmethylated_CpG_count}));
|
|
|
485 }
|
|
|
486
|
|
|
487 ### printing methylated CpG percentage if applicable
|
|
|
488 if ($percent_meCpG){
|
|
|
489 warn "C methylated in CpG context:\t${percent_meCpG}%\n";
|
|
|
490 print REPORT "C methylated in CpG context:\t${percent_meCpG}%\n";
|
|
|
491 }
|
|
|
492 else{
|
|
|
493 warn "Can't determine percentage of methylated Cs in CpG context if value was 0\n";
|
|
|
494 print REPORT "Can't determine percentage of methylated Cs in CpG context if value was 0\n";
|
|
|
495 }
|
|
|
496
|
|
|
497 ### printing methylated C percentage (CHG context) if applicable
|
|
|
498 if ($percent_meCHG){
|
|
|
499 warn "C methylated in CHG context:\t${percent_meCHG}%\n";
|
|
|
500 print REPORT "C methylated in CHG context:\t${percent_meCHG}%\n";
|
|
|
501 }
|
|
|
502 else{
|
|
|
503 warn "Can't determine percentage of methylated Cs in CHG context if value was 0\n";
|
|
|
504 print REPORT "Can't determine percentage of methylated Cs in CHG context if value was 0\n";
|
|
|
505 }
|
|
|
506
|
|
|
507 ### printing methylated C percentage (CHH context) if applicable
|
|
|
508 if ($percent_meCHH){
|
|
|
509 warn "C methylated in CHH context:\t${percent_meCHH}%\n\n\n";
|
|
|
510 print REPORT "C methylated in CHH context:\t${percent_meCHH}%\n\n\n";
|
|
|
511 }
|
|
|
512 else{
|
|
|
513 warn "Can't determine percentage of methylated Cs in CHH context if value was 0\n\n\n";
|
|
|
514 print REPORT "Can't determine percentage of methylated Cs in CHH context if value was 0\n\n\n";
|
|
|
515 }
|
|
|
516
|
|
|
517 if ($seqID_contains_tabs){
|
|
|
518 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";
|
|
|
519 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";
|
|
|
520 }
|
|
|
521 }
|
|
|
522
|
|
|
523 sub print_final_analysis_report_paired_ends{
|
|
|
524 my ($C_to_T_infile_1,$G_to_A_infile_1,$C_to_T_infile_2,$G_to_A_infile_2) = @_;
|
|
|
525 ### All sequences from the original sequence file have been analysed now, therefore deleting temporary C->T or G->A infiles
|
|
|
526 if ($directional){
|
|
|
527 my $deletion_successful = unlink "$temp_dir$C_to_T_infile_1","$temp_dir$G_to_A_infile_2";
|
|
|
528 if ($deletion_successful == 2){
|
|
|
529 warn "\nSuccessfully deleted the temporary files $temp_dir$C_to_T_infile_1 and $temp_dir$G_to_A_infile_2\n\n";
|
|
|
530 }
|
|
|
531 else{
|
|
|
532 warn "Could not delete temporary files $temp_dir$C_to_T_infile_1 and $temp_dir$G_to_A_infile_2 properly: $!\n";
|
|
|
533 }
|
|
|
534 }
|
|
|
535 else{
|
|
|
536 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$G_to_A_infile_2";
|
|
|
537 if ($deletion_successful == 4){
|
|
|
538 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";
|
|
|
539 }
|
|
|
540 else{
|
|
|
541 warn "Could not delete temporary files properly: $!\n";
|
|
|
542 }
|
|
|
543 }
|
|
|
544
|
|
|
545 ### printing a final report for the alignment procedure
|
|
|
546 warn "Final Alignment report\n",'='x22,"\n";
|
|
|
547 print REPORT "Final Alignment report\n",'='x22,"\n";
|
|
|
548 # foreach my $index (0..$#fhs){
|
|
|
549 # print "$fhs[$index]->{name}\n";
|
|
|
550 # print "$fhs[$index]->{seen}\talignments on the correct strand in total\n";
|
|
|
551 # print "$fhs[$index]->{wrong_strand}\talignments were discarded (nonsensical alignments)\n\n";
|
|
|
552 # }
|
|
|
553
|
|
|
554 ### printing a final report for the methylation call procedure
|
|
|
555 warn "Sequence pairs analysed in total:\t$counting{sequences_count}\n";
|
|
|
556 print REPORT "Sequence pairs analysed in total:\t$counting{sequences_count}\n";
|
|
|
557
|
|
|
558 my $percent_alignable_sequence_pairs = sprintf ("%.1f",$counting{unique_best_alignment_count}*100/$counting{sequences_count});
|
|
|
559 print "Number of paired-end alignments with a unique best hit:\t$counting{unique_best_alignment_count}\nMapping efficiency:\t${percent_alignable_sequence_pairs}%\n\n";
|
|
|
560 print REPORT "Number of paired-end alignments with a unique best hit:\t$counting{unique_best_alignment_count}\nMapping efficiency:\t${percent_alignable_sequence_pairs}% \n";
|
|
|
561
|
|
|
562 print "Sequence pairs with no alignments under any condition:\t$counting{no_single_alignment_found}\n";
|
|
|
563 print "Sequence pairs did not map uniquely:\t$counting{unsuitable_sequence_count}\n";
|
|
|
564 print "Sequence pairs which were discarded because genomic sequence could not be extracted:\t$counting{genomic_sequence_could_not_be_extracted_count}\n\n";
|
|
|
565 print "Number of sequence pairs with unique best (first) alignment came from the bowtie output:\n";
|
|
|
566 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";
|
|
|
567
|
|
|
568
|
|
|
569 print REPORT "Sequence pairs with no alignments under any condition:\t$counting{no_single_alignment_found}\n";
|
|
|
570 print REPORT "Sequence pairs did not map uniquely:\t$counting{unsuitable_sequence_count}\n";
|
|
|
571 print REPORT "Sequence pairs which were discarded because genomic sequence could not be extracted:\t$counting{genomic_sequence_could_not_be_extracted_count}\n\n";
|
|
|
572 print REPORT "Number of sequence pairs with unique best (first) alignment came from the bowtie output:\n";
|
|
|
573 print REPORT join ("\n","CT/GA/CT:\t$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$counting{CT_GA_GA_count}\t((converted) bottom strand)"),"\n\n";
|
|
|
574 ### detailed information about Cs analysed
|
|
|
575
|
|
|
576 if ($directional){
|
|
|
577 print "Number of alignments to (merely theoretical) complementary strands being rejected in total:\t$counting{alignments_rejected_count}\n\n";
|
|
|
578 print REPORT "Number of alignments to (merely theoretical) complementary strands being rejected in total:\t$counting{alignments_rejected_count}\n\n";
|
|
|
579 }
|
|
|
580
|
|
|
581 warn "Final Cytosine Methylation Report\n",'='x33,"\n";
|
|
|
582 print REPORT "Final Cytosine Methylation Report\n",'='x33,"\n";
|
|
|
583
|
|
|
584 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}+$counting{total_unmethylated_CpG_count};
|
|
|
585 warn "Total number of C's analysed:\t$total_number_of_C\n\n";
|
|
|
586 warn "Total methylated C's in CpG context:\t$counting{total_meCpG_count}\n";
|
|
|
587 warn "Total methylated C's in CHG context:\t$counting{total_meCHG_count}\n";
|
|
|
588 warn "Total methylated C's in CHH context:\t$counting{total_meCHH_count}\n\n";
|
|
|
589 warn "Total C to T conversions in CpG context:\t$counting{total_unmethylated_CpG_count}\n";
|
|
|
590 warn "Total C to T conversions in CHG context:\t$counting{total_unmethylated_CHG_count}\n";
|
|
|
591 warn "Total C to T conversions in CHH context:\t$counting{total_unmethylated_CHH_count}\n\n";
|
|
|
592
|
|
|
593 print REPORT "Total number of C's analysed:\t$total_number_of_C\n\n";
|
|
|
594 print REPORT "Total methylated C's in CpG context:\t$counting{total_meCpG_count}\n";
|
|
|
595 print REPORT "Total methylated C's in CHG context:\t$counting{total_meCHG_count}\n";
|
|
|
596 print REPORT "Total methylated C's in CHH context:\t$counting{total_meCHH_count}\n\n";
|
|
|
597 print REPORT "Total C to T conversions in CpG context:\t$counting{total_unmethylated_CpG_count}\n";
|
|
|
598 print REPORT "Total C to T conversions in CHG context:\t$counting{total_unmethylated_CHG_count}\n";
|
|
|
599 print REPORT "Total C to T conversions in CHH context:\t$counting{total_unmethylated_CHH_count}\n\n";
|
|
|
600
|
|
|
601 my $percent_meCHG;
|
|
|
602 if (($counting{total_meCHG_count}+$counting{total_unmethylated_CHG_count}) > 0){
|
|
|
603 $percent_meCHG = sprintf("%.1f",100*$counting{total_meCHG_count}/($counting{total_meCHG_count}+$counting{total_unmethylated_CHG_count}));
|
|
|
604 }
|
|
|
605
|
|
|
606 my $percent_meCHH;
|
|
|
607 if (($counting{total_meCHH_count}+$counting{total_unmethylated_CHH_count}) > 0){
|
|
|
608 $percent_meCHH = sprintf("%.1f",100*$counting{total_meCHH_count}/($counting{total_meCHH_count}+$counting{total_unmethylated_CHH_count}));
|
|
|
609 }
|
|
|
610
|
|
|
611 my $percent_meCpG;
|
|
|
612 if (($counting{total_meCpG_count}+$counting{total_unmethylated_CpG_count}) > 0){
|
|
|
613 $percent_meCpG = sprintf("%.1f",100*$counting{total_meCpG_count}/($counting{total_meCpG_count}+$counting{total_unmethylated_CpG_count}));
|
|
|
614 }
|
|
|
615
|
|
|
616 ### printing methylated CpG percentage if applicable
|
|
|
617 if ($percent_meCpG){
|
|
|
618 warn "C methylated in CpG context:\t${percent_meCpG}%\n";
|
|
|
619 print REPORT "C methylated in CpG context:\t${percent_meCpG}%\n";
|
|
|
620 }
|
|
|
621 else{
|
|
|
622 warn "Can't determine percentage of methylated Cs in CpG context if value was 0\n";
|
|
|
623 print REPORT "Can't determine percentage of methylated Cs in CpG context if value was 0\n";
|
|
|
624 }
|
|
|
625
|
|
|
626 ### printing methylated C percentage in CHG context if applicable
|
|
|
627 if ($percent_meCHG){
|
|
|
628 warn "C methylated in CHG context:\t${percent_meCHG}%\n";
|
|
|
629 print REPORT "C methylated in CHG context:\t${percent_meCHG}%\n";
|
|
|
630 }
|
|
|
631 else{
|
|
|
632 warn "Can't determine percentage of methylated Cs in CHG context if value was 0\n";
|
|
|
633 print REPORT "Can't determine percentage of methylated Cs in CHG context if value was 0\n";
|
|
|
634 }
|
|
|
635
|
|
|
636 ### printing methylated C percentage in CHH context if applicable
|
|
|
637 if ($percent_meCHH){
|
|
|
638 warn "C methylated in CHH context:\t${percent_meCHH}%\n\n\n";
|
|
|
639 print REPORT "C methylated in CHH context:\t${percent_meCHH}%\n\n\n";
|
|
|
640 }
|
|
|
641 else{
|
|
|
642 warn "Can't determine percentage of methylated Cs in CHH context if value was 0\n\n\n";
|
|
|
643 print REPORT "Can't determine percentage of methylated Cs in CHH context if value was 0\n\n\n";
|
|
|
644 }
|
|
|
645
|
|
|
646 }
|
|
|
647
|
|
|
648 sub process_single_end_fastA_file_for_methylation_call{
|
|
|
649 my ($sequence_file,$C_to_T_infile,$G_to_A_infile) = @_;
|
|
|
650 ### 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.
|
|
|
651 ### 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
|
|
|
652 ### the C->T or G->A version
|
|
|
653
|
|
|
654 ### gzipped version of the infile
|
|
|
655 if ($sequence_file =~ /\.gz$/){
|
|
|
656 open (IN,"zcat $sequence_file |") or die $!;
|
|
|
657 }
|
|
|
658 else{
|
|
|
659 open (IN,$sequence_file) or die $!;
|
|
|
660 }
|
|
|
661
|
|
|
662 my $count = 0;
|
|
|
663
|
|
|
664 warn "\nReading in the sequence file $sequence_file\n";
|
|
|
665 while (1) {
|
|
|
666 # last if ($counting{sequences_count} > 100);
|
|
|
667 my $identifier = <IN>;
|
|
|
668 my $sequence = <IN>;
|
|
|
669 last unless ($identifier and $sequence);
|
|
|
670
|
|
|
671 $identifier = fix_IDs($identifier); # this is to avoid problems with truncated read ID when they contain white spaces
|
|
|
672
|
|
|
673 ++$count;
|
|
|
674
|
|
|
675 if ($skip){
|
|
|
676 next unless ($count > $skip);
|
|
|
677 }
|
|
|
678 if ($upto){
|
|
|
679 last if ($count > $upto);
|
|
|
680 }
|
|
|
681
|
|
|
682 $counting{sequences_count}++;
|
|
|
683 if ($counting{sequences_count}%100000==0) {
|
|
|
684 warn "Processed $counting{sequences_count} sequences so far\n";
|
|
|
685 }
|
|
|
686 chomp $sequence;
|
|
|
687 chomp $identifier;
|
|
|
688
|
|
|
689 $identifier =~ s/^>//; # deletes the > at the beginning of FastA headers
|
|
|
690
|
|
|
691 my $return;
|
|
|
692 if ($bowtie2){
|
|
|
693 $return = check_bowtie_results_single_end_bowtie2 (uc$sequence,$identifier);
|
|
|
694 }
|
|
|
695 else{
|
|
|
696 $return = check_bowtie_results_single_end(uc$sequence,$identifier); # default Bowtie 1
|
|
|
697 }
|
|
|
698
|
|
|
699 unless ($return){
|
|
|
700 $return = 0;
|
|
|
701 }
|
|
|
702
|
|
|
703 # print the sequence to ambiguous.out if --ambiguous was specified
|
|
|
704 if ($ambiguous and $return == 2){
|
|
|
705 print AMBIG ">$identifier\n";
|
|
|
706 print AMBIG "$sequence\n";
|
|
|
707 }
|
|
|
708
|
|
|
709 # print the sequence to <unmapped.out> file if --un was specified
|
|
|
710 elsif ($unmapped and $return == 1){
|
|
|
711 print UNMAPPED ">$identifier\n";
|
|
|
712 print UNMAPPED "$sequence\n";
|
|
|
713 }
|
|
|
714 }
|
|
|
715 print "Processed $counting{sequences_count} sequences in total\n\n";
|
|
|
716
|
|
|
717 print_final_analysis_report_single_end($C_to_T_infile,$G_to_A_infile);
|
|
|
718
|
|
|
719 }
|
|
|
720
|
|
|
721 sub process_single_end_fastQ_file_for_methylation_call{
|
|
|
722 my ($sequence_file,$C_to_T_infile,$G_to_A_infile) = @_;
|
|
|
723 ### 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.
|
|
|
724 ### 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
|
|
|
725 ### the C->T or G->A version
|
|
|
726
|
|
|
727 ### gzipped version of the infile
|
|
|
728 if ($sequence_file =~ /\.gz$/){
|
|
|
729 open (IN,"zcat $sequence_file |") or die $!;
|
|
|
730 }
|
|
|
731 else{
|
|
|
732 open (IN,$sequence_file) or die $!;
|
|
|
733 }
|
|
|
734
|
|
|
735 my $count = 0;
|
|
|
736
|
|
|
737 warn "\nReading in the sequence file $sequence_file\n";
|
|
|
738 while (1) {
|
|
|
739 my $identifier = <IN>;
|
|
|
740 my $sequence = <IN>;
|
|
|
741 my $identifier_2 = <IN>;
|
|
|
742 my $quality_value = <IN>;
|
|
|
743 last unless ($identifier and $sequence and $identifier_2 and $quality_value);
|
|
|
744
|
|
|
745 $identifier = fix_IDs($identifier); # this is to avoid problems with truncated read ID when they contain white spaces
|
|
|
746
|
|
|
747 ++$count;
|
|
|
748
|
|
|
749 if ($skip){
|
|
|
750 next unless ($count > $skip);
|
|
|
751 }
|
|
|
752 if ($upto){
|
|
|
753 last if ($count > $upto);
|
|
|
754 }
|
|
|
755
|
|
|
756 $counting{sequences_count}++;
|
|
|
757
|
|
|
758 if ($counting{sequences_count}%1000000==0) {
|
|
|
759 warn "Processed $counting{sequences_count} sequences so far\n";
|
|
|
760 }
|
|
|
761 chomp $sequence;
|
|
|
762 chomp $identifier;
|
|
|
763 chomp $quality_value;
|
|
|
764
|
|
|
765 $identifier =~ s/^\@//; # deletes the @ at the beginning of Illumin FastQ headers
|
|
|
766
|
|
|
767 my $return;
|
|
|
768 if ($bowtie2){
|
|
|
769 $return = check_bowtie_results_single_end_bowtie2 (uc$sequence,$identifier,$quality_value);
|
|
|
770 }
|
|
|
771 else{
|
|
|
772 $return = check_bowtie_results_single_end(uc$sequence,$identifier,$quality_value); # default Bowtie 1
|
|
|
773 }
|
|
|
774
|
|
|
775 unless ($return){
|
|
|
776 $return = 0;
|
|
|
777 }
|
|
|
778
|
|
|
779 # print the sequence to ambiguous.out if --ambiguous was specified
|
|
|
780 if ($ambiguous and $return == 2){
|
|
|
781 print AMBIG "\@$identifier\n";
|
|
|
782 print AMBIG "$sequence\n";
|
|
|
783 print AMBIG $identifier_2;
|
|
|
784 print AMBIG "$quality_value\n";
|
|
|
785 }
|
|
|
786
|
|
|
787 # print the sequence to <unmapped.out> file if --un was specified
|
|
|
788 elsif ($unmapped and $return == 1){
|
|
|
789 print UNMAPPED "\@$identifier\n";
|
|
|
790 print UNMAPPED "$sequence\n";
|
|
|
791 print UNMAPPED $identifier_2;
|
|
|
792 print UNMAPPED "$quality_value\n";
|
|
|
793 }
|
|
|
794 }
|
|
|
795 print "Processed $counting{sequences_count} sequences in total\n\n";
|
|
|
796
|
|
|
797 print_final_analysis_report_single_end($C_to_T_infile,$G_to_A_infile);
|
|
|
798
|
|
|
799 }
|
|
|
800
|
|
|
801 sub process_fastA_files_for_paired_end_methylation_calls{
|
|
|
802 my ($sequence_file_1,$sequence_file_2,$C_to_T_infile_1,$G_to_A_infile_1,$C_to_T_infile_2,$G_to_A_infile_2) = @_;
|
|
|
803 ### Processing the two FastA sequence files; we need the actual sequences of both reads to compare them against the genomic sequence in order to
|
|
|
804 ### make a methylation call. The sequence idetifier per definition needs to be the same for a sequence pair used for paired-end mapping.
|
|
|
805 ### Now reading in the sequence files sequence by sequence and see if the current sequences produced an alignment to one (or both) of the
|
|
|
806 ### converted genomes (either the C->T or G->A version)
|
|
|
807
|
|
|
808 ### gzipped version of the infiles
|
|
|
809 if ($sequence_file_1 =~ /\.gz$/ and $sequence_file_2 =~ /\.gz$/){
|
|
|
810 open (IN1,"zcat $sequence_file_1 |") or die "Failed to open zcat pipe to $sequence_file_1 $!\n";
|
|
|
811 open (IN2,"zcat $sequence_file_2 |") or die "Failed to open zcat pipe to $sequence_file_2 $!\n";
|
|
|
812 }
|
|
|
813 else{
|
|
|
814 open (IN1,$sequence_file_1) or die $!;
|
|
|
815 open (IN2,$sequence_file_2) or die $!;
|
|
|
816 }
|
|
|
817
|
|
|
818 warn "\nReading in the sequence files $sequence_file_1 and $sequence_file_2\n";
|
|
|
819 ### Both files are required to have the exact same number of sequences, therefore we can process the sequences jointly one by one
|
|
|
820
|
|
|
821 my $count = 0;
|
|
|
822
|
|
|
823 while (1) {
|
|
|
824 # reading from the first input file
|
|
|
825 my $identifier_1 = <IN1>;
|
|
|
826 my $sequence_1 = <IN1>;
|
|
|
827 # reading from the second input file
|
|
|
828 my $identifier_2 = <IN2>;
|
|
|
829 my $sequence_2 = <IN2>;
|
|
|
830 last unless ($identifier_1 and $sequence_1 and $identifier_2 and $sequence_2);
|
|
|
831
|
|
|
832 $identifier_1 = fix_IDs($identifier_1); # this is to avoid problems with truncated read ID when they contain white spaces
|
|
|
833 $identifier_2 = fix_IDs($identifier_2);
|
|
|
834
|
|
|
835 ++$count;
|
|
|
836
|
|
|
837 if ($skip){
|
|
|
838 next unless ($count > $skip);
|
|
|
839 }
|
|
|
840 if ($upto){
|
|
|
841 last if ($count > $upto);
|
|
|
842 }
|
|
|
843
|
|
|
844 $counting{sequences_count}++;
|
|
|
845 if ($counting{sequences_count}%100000==0) {
|
|
|
846 warn "Processed $counting{sequences_count} sequences so far\n";
|
|
|
847 }
|
|
|
848 my $orig_identifier_1 = $identifier_1;
|
|
|
849 my $orig_identifier_2 = $identifier_2;
|
|
|
850
|
|
|
851 chomp $sequence_1;
|
|
|
852 chomp $identifier_1;
|
|
|
853 chomp $sequence_2;
|
|
|
854 chomp $identifier_2;
|
|
|
855
|
|
|
856 $identifier_1 =~ s/^>//; # deletes the > at the beginning of FastA headers
|
|
|
857
|
|
|
858 my $return;
|
|
|
859 if ($bowtie2){
|
|
|
860 $return = check_bowtie_results_paired_ends_bowtie2 (uc$sequence_1,uc$sequence_2,$identifier_1);
|
|
|
861 }
|
|
|
862 else{
|
|
|
863 $return = check_bowtie_results_paired_ends (uc$sequence_1,uc$sequence_2,$identifier_1);
|
|
|
864 }
|
|
|
865
|
|
|
866 unless ($return){
|
|
|
867 $return = 0;
|
|
|
868 }
|
|
|
869
|
|
|
870 # print the sequences to ambiguous_1 and _2 if --ambiguous was specified
|
|
|
871 if ($ambiguous and $return == 2){
|
|
|
872 print AMBIG_1 $orig_identifier_1;
|
|
|
873 print AMBIG_1 "$sequence_1\n";
|
|
|
874 print AMBIG_2 $orig_identifier_2;
|
|
|
875 print AMBIG_2 "$sequence_2\n";
|
|
|
876 }
|
|
|
877
|
|
|
878 # print the sequences to unmapped_1.out and unmapped_2.out if --un was specified
|
|
|
879 elsif ($unmapped and $return == 1){
|
|
|
880 print UNMAPPED_1 $orig_identifier_1;
|
|
|
881 print UNMAPPED_1 "$sequence_1\n";
|
|
|
882 print UNMAPPED_2 $orig_identifier_2;
|
|
|
883 print UNMAPPED_2 "$sequence_2\n";
|
|
|
884 }
|
|
|
885 }
|
|
|
886
|
|
|
887 print "Processed $counting{sequences_count} sequences in total\n\n";
|
|
|
888
|
|
|
889 print_final_analysis_report_paired_ends($C_to_T_infile_1,$G_to_A_infile_1,$C_to_T_infile_2,$G_to_A_infile_2);
|
|
|
890
|
|
|
891 }
|
|
|
892
|
|
|
893 sub process_fastQ_files_for_paired_end_methylation_calls{
|
|
|
894 my ($sequence_file_1,$sequence_file_2,$C_to_T_infile_1,$G_to_A_infile_1,$C_to_T_infile_2,$G_to_A_infile_2) = @_;
|
|
|
895 ### Processing the two Illumina sequence files; we need the actual sequence of both reads to compare them against the genomic sequence in order to
|
|
|
896 ### make a methylation call. The sequence identifier per definition needs to be same for a sequence pair used for paired-end alignments.
|
|
|
897 ### Now reading in the sequence files sequence by sequence and see if the current sequences produced a paired-end alignment to one (or both)
|
|
|
898 ### of the converted genomes (either C->T or G->A version)
|
|
|
899
|
|
|
900 ### gzipped version of the infiles
|
|
|
901 if ($sequence_file_1 =~ /\.gz$/ and $sequence_file_2 =~ /\.gz$/){
|
|
|
902 open (IN1,"zcat $sequence_file_1 |") or die "Failed to open zcat pipe to $sequence_file_1 $!\n";
|
|
|
903 open (IN2,"zcat $sequence_file_2 |") or die "Failed to open zcat pipe to $sequence_file_2 $!\n";
|
|
|
904 }
|
|
|
905 else{
|
|
|
906 open (IN1,$sequence_file_1) or die $!;
|
|
|
907 open (IN2,$sequence_file_2) or die $!;
|
|
|
908 }
|
|
|
909
|
|
|
910 my $count = 0;
|
|
|
911
|
|
|
912 warn "\nReading in the sequence files $sequence_file_1 and $sequence_file_2\n";
|
|
|
913 ### Both files are required to have the exact same number of sequences, therefore we can process the sequences jointly one by one
|
|
|
914 while (1) {
|
|
|
915 # reading from the first input file
|
|
|
916 my $identifier_1 = <IN1>;
|
|
|
917 my $sequence_1 = <IN1>;
|
|
|
918 my $ident_1 = <IN1>; # not needed
|
|
|
919 my $quality_value_1 = <IN1>; # not needed
|
|
|
920 # reading from the second input file
|
|
|
921 my $identifier_2 = <IN2>;
|
|
|
922 my $sequence_2 = <IN2>;
|
|
|
923 my $ident_2 = <IN2>; # not needed
|
|
|
924 my $quality_value_2 = <IN2>; # not needed
|
|
|
925 last unless ($identifier_1 and $sequence_1 and $quality_value_1 and $identifier_2 and $sequence_2 and $quality_value_2);
|
|
|
926
|
|
|
927 $identifier_1 = fix_IDs($identifier_1); # this is to avoid problems with truncated read ID when they contain white spaces
|
|
|
928 $identifier_2 = fix_IDs($identifier_2);
|
|
|
929
|
|
|
930 ++$count;
|
|
|
931
|
|
|
932 if ($skip){
|
|
|
933 next unless ($count > $skip);
|
|
|
934 }
|
|
|
935 if ($upto){
|
|
|
936 last if ($count > $upto);
|
|
|
937 }
|
|
|
938
|
|
|
939 $counting{sequences_count}++;
|
|
|
940 if ($counting{sequences_count}%100000==0) {
|
|
|
941 warn "Processed $counting{sequences_count} sequences so far\n";
|
|
|
942 }
|
|
|
943
|
|
|
944 my $orig_identifier_1 = $identifier_1;
|
|
|
945 my $orig_identifier_2 = $identifier_2;
|
|
|
946
|
|
|
947 chomp $sequence_1;
|
|
|
948 chomp $identifier_1;
|
|
|
949 chomp $sequence_2;
|
|
|
950 chomp $identifier_2;
|
|
|
951 chomp $quality_value_1;
|
|
|
952 chomp $quality_value_2;
|
|
|
953
|
|
|
954 $identifier_1 =~ s/^\@//; # deletes the @ at the beginning of the FastQ ID
|
|
|
955
|
|
|
956 my $return;
|
|
|
957 if ($bowtie2){
|
|
|
958 $return = check_bowtie_results_paired_ends_bowtie2 (uc$sequence_1,uc$sequence_2,$identifier_1,$quality_value_1,$quality_value_2);
|
|
|
959 }
|
|
|
960 else{
|
|
|
961 $return = check_bowtie_results_paired_ends (uc$sequence_1,uc$sequence_2,$identifier_1,$quality_value_1,$quality_value_2);
|
|
|
962 }
|
|
|
963
|
|
|
964 unless ($return){
|
|
|
965 $return = 0;
|
|
|
966 }
|
|
|
967
|
|
|
968 # print the sequences to ambiguous_1 and _2 if --ambiguous was specified
|
|
|
969 if ($ambiguous and $return == 2){
|
|
|
970 # seq_1
|
|
|
971 print AMBIG_1 $orig_identifier_1;
|
|
|
972 print AMBIG_1 "$sequence_1\n";
|
|
|
973 print AMBIG_1 $ident_1;
|
|
|
974 print AMBIG_1 "$quality_value_1\n";
|
|
|
975 # seq_2
|
|
|
976 print AMBIG_2 $orig_identifier_2;
|
|
|
977 print AMBIG_2 "$sequence_2\n";
|
|
|
978 print AMBIG_2 $ident_2;
|
|
|
979 print AMBIG_2 "$quality_value_2\n";
|
|
|
980 }
|
|
|
981
|
|
|
982 # print the sequences to unmapped_1.out and unmapped_2.out if --un was specified
|
|
|
983 elsif ($unmapped and $return == 1){
|
|
|
984 # seq_1
|
|
|
985 print UNMAPPED_1 $orig_identifier_1;
|
|
|
986 print UNMAPPED_1 "$sequence_1\n";
|
|
|
987 print UNMAPPED_1 $ident_1;
|
|
|
988 print UNMAPPED_1 "$quality_value_1\n";
|
|
|
989 # seq_2
|
|
|
990 print UNMAPPED_2 $orig_identifier_2;
|
|
|
991 print UNMAPPED_2 "$sequence_2\n";
|
|
|
992 print UNMAPPED_2 $ident_2;
|
|
|
993 print UNMAPPED_2 "$quality_value_2\n";
|
|
|
994 }
|
|
|
995 }
|
|
|
996
|
|
|
997 print "Processed $counting{sequences_count} sequences in total\n\n";
|
|
|
998
|
|
|
999 print_final_analysis_report_paired_ends($C_to_T_infile_1,$G_to_A_infile_1,$C_to_T_infile_2,$G_to_A_infile_2);
|
|
|
1000
|
|
|
1001 }
|
|
|
1002
|
|
|
1003 sub check_bowtie_results_single_end{
|
|
|
1004 my ($sequence,$identifier,$quality_value) = @_;
|
|
|
1005
|
|
|
1006 unless ($quality_value){ # FastA sequences get assigned a quality value of Phred 40 throughout
|
|
|
1007 $quality_value = 'I'x(length$sequence);
|
|
|
1008 }
|
|
|
1009
|
|
|
1010 my %mismatches = ();
|
|
|
1011 ### reading from the bowtie output files to see if this sequence aligned to a bisulfite converted genome
|
|
|
1012 foreach my $index (0..$#fhs){
|
|
|
1013
|
|
|
1014 ### skipping this index if the last alignment has been set to undefined already (i.e. end of bowtie output)
|
|
|
1015 next unless ($fhs[$index]->{last_line} and defined $fhs[$index]->{last_seq_id});
|
|
|
1016 ### if the sequence we are currently looking at produced an alignment we are doing various things with it
|
|
|
1017 if ($fhs[$index]->{last_seq_id} eq $identifier) {
|
|
|
1018 ###############################################################
|
|
|
1019 ### STEP I Now processing the alignment stored in last_line ###
|
|
|
1020 ###############################################################
|
|
|
1021 my $valid_alignment_found_1 = decide_whether_single_end_alignment_is_valid($index,$identifier);
|
|
|
1022 ### sequences can fail at this point if there was only 1 seq in the wrong orientation, or if there were 2 seqs, both in the wrong orientation
|
|
|
1023 ### we only continue to extract useful information about this alignment if 1 was returned
|
|
|
1024 if ($valid_alignment_found_1 == 1){
|
|
|
1025 ### Bowtie outputs which made it this far are in the correct orientation, so we can continue to analyse the alignment itself
|
|
|
1026 ### need to extract the chromosome number from the bowtie output (which is either XY_cf (complete forward) or XY_cr (complete reverse)
|
|
|
1027 my ($id,$strand,$mapped_chromosome,$position,$bowtie_sequence,$mismatch_info) = (split (/\t/,$fhs[$index]->{last_line},-1))[0,1,2,3,4,7];
|
|
|
1028
|
|
|
1029 unless($mismatch_info){
|
|
|
1030 $mismatch_info = '';
|
|
|
1031 }
|
|
|
1032
|
|
|
1033 chomp $mismatch_info;
|
|
|
1034 my $chromosome;
|
|
|
1035 if ($mapped_chromosome =~ s/_(CT|GA)_converted$//){
|
|
|
1036 $chromosome = $mapped_chromosome;
|
|
|
1037 }
|
|
|
1038 else{
|
|
|
1039 die "Chromosome number extraction failed for $mapped_chromosome\n";
|
|
|
1040 }
|
|
|
1041 ### Now extracting the number of mismatches to the converted genome
|
|
|
1042 my $number_of_mismatches;
|
|
|
1043 if ($mismatch_info eq ''){
|
|
|
1044 $number_of_mismatches = 0;
|
|
|
1045 }
|
|
|
1046 elsif ($mismatch_info =~ /^\d/){
|
|
|
1047 my @mismatches = split (/,/,$mismatch_info);
|
|
|
1048 $number_of_mismatches = scalar @mismatches;
|
|
|
1049 }
|
|
|
1050 else{
|
|
|
1051 die "Something weird is going on with the mismatch field:\t>>> $mismatch_info <<<\n";
|
|
|
1052 }
|
|
|
1053 ### creating a composite location variable from $chromosome and $position and storing the alignment information in a temporary hash table
|
|
|
1054 my $alignment_location = join (":",$chromosome,$position);
|
|
|
1055 ### 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
|
|
|
1056 ### strand) were methylated and therefore protected. It is not needed to overwrite the same positional entry with a second entry for the same
|
|
|
1057 ### location (the genomic sequence extraction and methylation would not be affected by this, only the thing which would change is the index
|
|
|
1058 ### number for the found alignment)
|
|
|
1059 unless (exists $mismatches{$number_of_mismatches}->{$alignment_location}){
|
|
|
1060 $mismatches{$number_of_mismatches}->{$alignment_location}->{seq_id}=$id;
|
|
|
1061 $mismatches{$number_of_mismatches}->{$alignment_location}->{bowtie_sequence}=$bowtie_sequence;
|
|
|
1062 $mismatches{$number_of_mismatches}->{$alignment_location}->{index}=$index;
|
|
|
1063 $mismatches{$number_of_mismatches}->{$alignment_location}->{chromosome}=$chromosome;
|
|
|
1064 $mismatches{$number_of_mismatches}->{$alignment_location}->{position}=$position;
|
|
|
1065 }
|
|
|
1066 $number_of_mismatches = undef;
|
|
|
1067 ##################################################################################################################################################
|
|
|
1068 ### 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
|
|
|
1069 ### 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
|
|
|
1070 ### be returned as $valid_alignment_found and it will then be processed in the next round only.
|
|
|
1071 ##################################################################################################################################################
|
|
|
1072 my $newline = $fhs[$index]->{fh}-> getline();
|
|
|
1073 if ($newline){
|
|
|
1074 my ($seq_id) = split (/\t/,$newline);
|
|
|
1075 $fhs[$index]->{last_seq_id} = $seq_id;
|
|
|
1076 $fhs[$index]->{last_line} = $newline;
|
|
|
1077 }
|
|
|
1078 else {
|
|
|
1079 # assigning undef to last_seq_id and last_line and jumping to the next index (end of bowtie output)
|
|
|
1080 $fhs[$index]->{last_seq_id} = undef;
|
|
|
1081 $fhs[$index]->{last_line} = undef;
|
|
|
1082 next;
|
|
|
1083 }
|
|
|
1084 my $valid_alignment_found_2 = decide_whether_single_end_alignment_is_valid($index,$identifier);
|
|
|
1085 ### we only continue to extract useful information about this second alignment if 1 was returned
|
|
|
1086 if ($valid_alignment_found_2 == 1){
|
|
|
1087 ### If the second Bowtie output made it this far it is in the correct orientation, so we can continue to analyse the alignment itself
|
|
|
1088 ### need to extract the chromosome number from the bowtie output (which is either XY_cf (complete forward) or XY_cr (complete reverse)
|
|
|
1089 my ($id,$strand,$mapped_chromosome,$position,$bowtie_sequence,$mismatch_info) = (split (/\t/,$fhs[$index]->{last_line},-1))[0,1,2,3,4,7];
|
|
|
1090 unless($mismatch_info){
|
|
|
1091 $mismatch_info = '';
|
|
|
1092 }
|
|
|
1093 chomp $mismatch_info;
|
|
|
1094
|
|
|
1095 my $chromosome;
|
|
|
1096 if ($mapped_chromosome =~ s/_(CT|GA)_converted$//){
|
|
|
1097 $chromosome = $mapped_chromosome;
|
|
|
1098 }
|
|
|
1099 else{
|
|
|
1100 die "Chromosome number extraction failed for $mapped_chromosome\n";
|
|
|
1101 }
|
|
|
1102
|
|
|
1103 ### Now extracting the number of mismatches to the converted genome
|
|
|
1104 my $number_of_mismatches;
|
|
|
1105 if ($mismatch_info eq ''){
|
|
|
1106 $number_of_mismatches = 0;
|
|
|
1107 }
|
|
|
1108 elsif ($mismatch_info =~ /^\d/){
|
|
|
1109 my @mismatches = split (/,/,$mismatch_info);
|
|
|
1110 $number_of_mismatches = scalar @mismatches;
|
|
|
1111 }
|
|
|
1112 else{
|
|
|
1113 die "Something weird is going on with the mismatch field\n";
|
|
|
1114 }
|
|
|
1115 ### creating a composite location variable from $chromosome and $position and storing the alignment information in a temporary hash table
|
|
|
1116 ### extracting the chromosome number from the bowtie output (see above)
|
|
|
1117 my $alignment_location = join (":",$chromosome,$position);
|
|
|
1118 ### In the special case that two differently converted sequences align against differently converted genomes, but to the same position
|
|
|
1119 ### with the same number of mismatches (or perfect matches), the chromosome, position and number of mismatches are the same. In this
|
|
|
1120 ### case we are not writing the same entry out a second time.
|
|
|
1121 unless (exists $mismatches{$number_of_mismatches}->{$alignment_location}){
|
|
|
1122 $mismatches{$number_of_mismatches}->{$alignment_location}->{seq_id}=$id;
|
|
|
1123 $mismatches{$number_of_mismatches}->{$alignment_location}->{bowtie_sequence}=$bowtie_sequence;
|
|
|
1124 $mismatches{$number_of_mismatches}->{$alignment_location}->{index}=$index;
|
|
|
1125 $mismatches{$number_of_mismatches}->{$alignment_location}->{chromosome}=$chromosome;
|
|
|
1126 $mismatches{$number_of_mismatches}->{$alignment_location}->{position}=$position;
|
|
|
1127 }
|
|
|
1128 ####################################################################################################################################
|
|
|
1129 #### STEP III Now reading in one more line which has to be the next alignment to be analysed. Adding it to @fhs ->{last_line} ###
|
|
|
1130 ####################################################################################################################################
|
|
|
1131 $newline = $fhs[$index]->{fh}-> getline();
|
|
|
1132 if ($newline){
|
|
|
1133 my ($seq_id) = split (/\t/,$newline);
|
|
|
1134 die "The same seq ID occurred more than twice in a row\n" if ($seq_id eq $identifier);
|
|
|
1135 $fhs[$index]->{last_seq_id} = $seq_id;
|
|
|
1136 $fhs[$index]->{last_line} = $newline;
|
|
|
1137 next;
|
|
|
1138 }
|
|
|
1139 else {
|
|
|
1140 # assigning undef to last_seq_id and last_line and jumping to the next index (end of bowtie output)
|
|
|
1141 $fhs[$index]->{last_seq_id} = undef;
|
|
|
1142 $fhs[$index]->{last_line} = undef;
|
|
|
1143 next;
|
|
|
1144 }
|
|
|
1145 ### still within the 2nd sequence in correct orientation found
|
|
|
1146 }
|
|
|
1147 ### still withing the 1st sequence in correct orientation found
|
|
|
1148 }
|
|
|
1149 ### still within the if (last_seq_id eq identifier) condition
|
|
|
1150 }
|
|
|
1151 ### still within foreach index loop
|
|
|
1152 }
|
|
|
1153 ### if there was not a single alignment found for a certain sequence we will continue with the next sequence in the sequence file
|
|
|
1154 unless(%mismatches){
|
|
|
1155 $counting{no_single_alignment_found}++;
|
|
|
1156 if ($unmapped){
|
|
|
1157 return 1; ### We will print this sequence out as unmapped sequence if --un unmapped.out has been specified
|
|
|
1158 }
|
|
|
1159 else{
|
|
|
1160 return;
|
|
|
1161 }
|
|
|
1162 }
|
|
|
1163 #######################################################################################################################################################
|
|
|
1164 #######################################################################################################################################################
|
|
|
1165 ### 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 ###
|
|
|
1166 ### 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 ###
|
|
|
1167 ### meth_call variables, if there are multiple hits with the same amount of (lowest) mismatches we are discarding the sequence altogether ###
|
|
|
1168 #######################################################################################################################################################
|
|
|
1169 #######################################################################################################################################################
|
|
|
1170 ### Going to use the variable $sequence_fails as a 'memory' if a sequence could not be aligned uniquely (set to 1 then)
|
|
|
1171 my $sequence_fails = 0;
|
|
|
1172 ### Declaring an empty hash reference which will store all information we need for the methylation call
|
|
|
1173 my $methylation_call_params; # hash reference!
|
|
|
1174 ### sorting in ascending order
|
|
|
1175 foreach my $mismatch_number (sort {$a<=>$b} keys %mismatches){
|
|
|
1176
|
|
|
1177 ### if there is only 1 entry in the hash with the lowest number of mismatches we accept it as the best alignment
|
|
|
1178 if (scalar keys %{$mismatches{$mismatch_number}} == 1){
|
|
|
1179 for my $unique_best_alignment (keys %{$mismatches{$mismatch_number}}){
|
|
|
1180 $methylation_call_params->{$identifier}->{bowtie_sequence} = $mismatches{$mismatch_number}->{$unique_best_alignment}->{bowtie_sequence};
|
|
|
1181 $methylation_call_params->{$identifier}->{chromosome} = $mismatches{$mismatch_number}->{$unique_best_alignment}->{chromosome};
|
|
|
1182 $methylation_call_params->{$identifier}->{position} = $mismatches{$mismatch_number}->{$unique_best_alignment}->{position};
|
|
|
1183 $methylation_call_params->{$identifier}->{index} = $mismatches{$mismatch_number}->{$unique_best_alignment}->{index};
|
|
|
1184 $methylation_call_params->{$identifier}->{number_of_mismatches} = $mismatch_number;
|
|
|
1185 }
|
|
|
1186 }
|
|
|
1187 elsif (scalar keys %{$mismatches{$mismatch_number}} == 3){
|
|
|
1188 ### 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
|
|
|
1189 ### come from different alignments processes (== indices) or (ii) one sequence alignment (== index) will give a unique best alignment, whereas a
|
|
|
1190 ### second one will produce 2 (or potentially many) alignments for the same sequence but in a different conversion state or against a different genome
|
|
|
1191 ### version (or both). This becomes especially relevant for highly converted sequences in which all Cs have been converted to Ts in the bisulfite
|
|
|
1192 ### reaction. E.g.
|
|
|
1193 ### CAGTCACGCGCGCGCG will become
|
|
|
1194 ### TAGTTATGTGTGTGTG in the CT transformed version, which will ideally still give the correct alignment in the CT->CT alignment condition.
|
|
|
1195 ### If the same read will then become G->A transformed as well however, the resulting sequence will look differently and potentially behave
|
|
|
1196 ### differently in a GA->GA alignment and this depends on the methylation state of the original sequence!:
|
|
|
1197 ### G->A conversion:
|
|
|
1198 ### highly methylated: CAATCACACACACACA
|
|
|
1199 ### highly converted : TAATTATATATATATA <== this sequence has a reduced complexity (only 2 bases left and not 3), and it is more likely to produce
|
|
|
1200 ### 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
|
|
|
1201 ### there will be 3 alignments with the same number of lowest mismatches!! This in turn means that highly methylated and thereby not converted
|
|
|
1202 ### sequences are more likely to pass the alignment step, thereby creating a bias for methylated reads compared to their non-methylated counterparts.
|
|
|
1203 ### We do not want any bias, whatsover. Therefore if we have 1 sequence producing a unique best alignment and the second and third conditions
|
|
|
1204 ### producing alignments only after performing an additional (theoretical) conversion we want to keep the best alignment with the lowest number of
|
|
|
1205 ### additional transliterations performed. Thus we want to have a look at the level of complexity of the sequences producing the alignment.
|
|
|
1206 ### In the above example the number of transliterations required to transform the actual sequence
|
|
|
1207 ### to the C->T version would be TAGTTATGTGTGTGTG -> TAGTTATGTGTGTGTG = 0; (assuming this gives the correct alignment)
|
|
|
1208 ### in the G->A case it would be TAGTTATGTGTGTGTG -> TAATTATATATATATA = 6; (assuming this gives multiple wrong alignments)
|
|
|
1209 ### if the sequence giving a unique best alignment required a lower number of transliterations than the second best sequence yielding alignments
|
|
|
1210 ### while requiring a much higher number of transliterations, we are going to accept the unique best alignment with the lowest number of performed
|
|
|
1211 ### 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
|
|
|
1212 ### smaller than the number of tranliterations of the second best sequence. Everything will be flagged with $sequence_fails = 1 and discarded.
|
|
|
1213 my @three_candidate_seqs;
|
|
|
1214 foreach my $composite_location (keys (%{$mismatches{$mismatch_number}}) ){
|
|
|
1215 my $transliterations_performed;
|
|
|
1216 if ($mismatches{$mismatch_number}->{$composite_location}->{index} == 0 or $mismatches{$mismatch_number}->{$composite_location}->{index} == 1){
|
|
|
1217 $transliterations_performed = determine_number_of_transliterations_performed($sequence,'CT');
|
|
|
1218 }
|
|
|
1219 elsif ($mismatches{$mismatch_number}->{$composite_location}->{index} == 2 or $mismatches{$mismatch_number}->{$composite_location}->{index} == 3){
|
|
|
1220 $transliterations_performed = determine_number_of_transliterations_performed($sequence,'GA');
|
|
|
1221 }
|
|
|
1222 else{
|
|
|
1223 die "unexpected index number range $!\n";
|
|
|
1224 }
|
|
|
1225 push @three_candidate_seqs,{
|
|
|
1226 index =>$mismatches{$mismatch_number}->{$composite_location}->{index},
|
|
|
1227 bowtie_sequence => $mismatches{$mismatch_number}->{$composite_location}->{bowtie_sequence},
|
|
|
1228 mismatch_number => $mismatch_number,
|
|
|
1229 chromosome => $mismatches{$mismatch_number}->{$composite_location}->{chromosome},
|
|
|
1230 position => $mismatches{$mismatch_number}->{$composite_location}->{position},
|
|
|
1231 seq_id => $mismatches{$mismatch_number}->{$composite_location}->{seq_id},
|
|
|
1232 transliterations_performed => $transliterations_performed,
|
|
|
1233 };
|
|
|
1234 }
|
|
|
1235 ### sorting in ascending order for the lowest number of transliterations performed
|
|
|
1236 @three_candidate_seqs = sort {$a->{transliterations_performed} <=> $b->{transliterations_performed}} @three_candidate_seqs;
|
|
|
1237 my $first_array_element = $three_candidate_seqs[0]->{transliterations_performed};
|
|
|
1238 my $second_array_element = $three_candidate_seqs[1]->{transliterations_performed};
|
|
|
1239 my $third_array_element = $three_candidate_seqs[2]->{transliterations_performed};
|
|
|
1240 # print "$first_array_element\t$second_array_element\t$third_array_element\n";
|
|
|
1241 if (($first_array_element*2) < $second_array_element){
|
|
|
1242 $counting{low_complexity_alignments_overruled_count}++;
|
|
|
1243 ### taking the index with the unique best hit and over ruling low complexity alignments with 2 hits
|
|
|
1244 $methylation_call_params->{$identifier}->{bowtie_sequence} = $three_candidate_seqs[0]->{bowtie_sequence};
|
|
|
1245 $methylation_call_params->{$identifier}->{chromosome} = $three_candidate_seqs[0]->{chromosome};
|
|
|
1246 $methylation_call_params->{$identifier}->{position} = $three_candidate_seqs[0]->{position};
|
|
|
1247 $methylation_call_params->{$identifier}->{index} = $three_candidate_seqs[0]->{index};
|
|
|
1248 $methylation_call_params->{$identifier}->{number_of_mismatches} = $mismatch_number;
|
|
|
1249 # print "Overruled low complexity alignments! Using $first_array_element and disregarding $second_array_element and $third_array_element\n";
|
|
|
1250 }
|
|
|
1251 else{
|
|
|
1252 $sequence_fails = 1;
|
|
|
1253 }
|
|
|
1254 }
|
|
|
1255 else{
|
|
|
1256 $sequence_fails = 1;
|
|
|
1257 }
|
|
|
1258 ### after processing the alignment with the lowest number of mismatches we exit
|
|
|
1259 last;
|
|
|
1260 }
|
|
|
1261 ### skipping the sequence completely if there were multiple alignments with the same amount of lowest mismatches found at different positions
|
|
|
1262 if ($sequence_fails == 1){
|
|
|
1263 $counting{unsuitable_sequence_count}++;
|
|
|
1264 if ($ambiguous){
|
|
|
1265 return 2; # => exits to next sequence, and prints it out to multiple_alignments.out if --ambiguous has been specified
|
|
|
1266 }
|
|
|
1267 if ($unmapped){
|
|
|
1268 return 1; # => exits to next sequence, and prints it out to unmapped.out if --un has been specified
|
|
|
1269 }
|
|
|
1270 else{
|
|
|
1271 return 0; # => exits to next sequence (default)
|
|
|
1272 }
|
|
|
1273 }
|
|
|
1274
|
|
|
1275 ### --DIRECTIONAL
|
|
|
1276 ### If the option --directional has been specified the user wants to consider only alignments to the original top strand or the original bottom strand. We will therefore
|
|
|
1277 ### discard all alignments to strands complementary to the original strands, as they should not exist in reality due to the library preparation protocol
|
|
|
1278 if ($directional){
|
|
|
1279 if ( ($methylation_call_params->{$identifier}->{index} == 2) or ($methylation_call_params->{$identifier}->{index} == 3) ){
|
|
|
1280 # warn "Alignment rejected! (index was: $methylation_call_params->{$identifier}->{index})\n";
|
|
|
1281 $counting{alignments_rejected_count}++;
|
|
|
1282 return 0;
|
|
|
1283 }
|
|
|
1284 }
|
|
|
1285
|
|
|
1286 ### If the sequence has not been rejected so far it will have a unique best alignment
|
|
|
1287 $counting{unique_best_alignment_count}++;
|
|
|
1288 extract_corresponding_genomic_sequence_single_end($identifier,$methylation_call_params);
|
|
|
1289 ### check test to see if the genomic sequence we extracted has the same length as the observed sequence+2, and only then we perform the methylation call
|
|
|
1290 if (length($methylation_call_params->{$identifier}->{unmodified_genomic_sequence}) != length($sequence)+2){
|
|
|
1291 warn "Chromosomal sequence could not be extracted for\t$identifier\t$methylation_call_params->{$identifier}->{chromosome}\t$methylation_call_params->{$identifier}->{position}\n";
|
|
|
1292 $counting{genomic_sequence_could_not_be_extracted_count}++;
|
|
|
1293 return 0;
|
|
|
1294 }
|
|
|
1295
|
|
|
1296 ### otherwise we are set to perform the actual methylation call
|
|
|
1297 $methylation_call_params->{$identifier}->{methylation_call} = methylation_call($identifier,$sequence,$methylation_call_params->{$identifier}->{unmodified_genomic_sequence},$methylation_call_params->{$identifier}->{read_conversion});
|
|
|
1298
|
|
|
1299 print_bisulfite_mapping_result_single_end($identifier,$sequence,$methylation_call_params,$quality_value);
|
|
|
1300 return 0; ## otherwise 1 will be returned by default, which would print the sequence to unmapped.out
|
|
|
1301 }
|
|
|
1302
|
|
|
1303 sub check_bowtie_results_single_end_bowtie2{
|
|
|
1304 my ($sequence,$identifier,$quality_value) = @_;
|
|
|
1305
|
|
|
1306 unless ($quality_value){ # FastA sequences get assigned a quality value of Phred 40 throughout
|
|
|
1307 $quality_value = 'I'x(length$sequence);
|
|
|
1308 }
|
|
|
1309
|
|
|
1310 # 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.
|
|
|
1311 # $identifier =~ s/\/[1234567890]+$//; # some sequencers don't just have /1 or /2 at the end of read IDs
|
|
|
1312
|
|
|
1313 my $alignment_ambiguous = 0;
|
|
|
1314
|
|
|
1315 my %alignments = ();
|
|
|
1316
|
|
|
1317 ### reading from the Bowtie 2 output filehandles
|
|
|
1318 foreach my $index (0..$#fhs){
|
|
|
1319 # print "Index: $index\n";
|
|
|
1320 # print "$fhs[$index]->{last_line}\n";
|
|
|
1321 # print "$fhs[$index]->{last_seq_id}\n\n";
|
|
|
1322
|
|
|
1323 ### skipping this index if the last alignment has been set to undefined already (i.e. end of bowtie output)
|
|
|
1324 next unless ($fhs[$index]->{last_line} and defined $fhs[$index]->{last_seq_id});
|
|
|
1325
|
|
|
1326 ### if the sequence we are currently looking at produced an alignment we are doing various things with it
|
|
|
1327 # print "last seq id: $fhs[$index]->{last_seq_id} and identifier: $identifier\n";
|
|
|
1328
|
|
|
1329 if ($fhs[$index]->{last_seq_id} eq $identifier) {
|
|
|
1330
|
|
|
1331 # SAM format specifications for Bowtie 2
|
|
|
1332 # (1) Name of read that aligned
|
|
|
1333 # (2) Sum of all applicable flags. Flags relevant to Bowtie are:
|
|
|
1334 # 1 The read is one of a pair
|
|
|
1335 # 2 The alignment is one end of a proper paired-end alignment
|
|
|
1336 # 4 The read has no reported alignments
|
|
|
1337 # 8 The read is one of a pair and has no reported alignments
|
|
|
1338 # 16 The alignment is to the reverse reference strand
|
|
|
1339 # 32 The other mate in the paired-end alignment is aligned to the reverse reference strand
|
|
|
1340 # 64 The read is mate 1 in a pair
|
|
|
1341 # 128 The read is mate 2 in a pair
|
|
|
1342 # 256 The read has multiple mapping states
|
|
|
1343 # (3) Name of reference sequence where alignment occurs (unmapped reads have a *)
|
|
|
1344 # (4) 1-based offset into the forward reference strand where leftmost character of the alignment occurs (0 for unmapped reads)
|
|
|
1345 # (5) Mapping quality (255 means MAPQ is not available)
|
|
|
1346 # (6) CIGAR string representation of alignment (* if unavailable)
|
|
|
1347 # (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.
|
|
|
1348 # (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.
|
|
|
1349 # (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.
|
|
|
1350 # (10) Read sequence (reverse-complemented if aligned to the reverse strand)
|
|
|
1351 # (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.
|
|
|
1352 # (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:
|
|
|
1353 # 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.
|
|
|
1354 # 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.
|
|
|
1355 # 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.
|
|
|
1356 # XN:i:<N> The number of ambiguous bases in the reference covering this alignment. Only present if SAM record is for an aligned read.
|
|
|
1357 # XM:i:<N> The number of mismatches in the alignment. Only present if SAM record is for an aligned read.
|
|
|
1358 # 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.
|
|
|
1359 # 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.
|
|
|
1360 # 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.
|
|
|
1361 # YF:Z:<N> String indicating reason why the read was filtered out. See also: Filtering. Only appears for reads that were filtered out.
|
|
|
1362 # 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.
|
|
|
1363
|
|
|
1364 my ($id,$flag,$mapped_chromosome,$position,$mapping_quality,$cigar,$bowtie_sequence,$qual) = (split (/\t/,$fhs[$index]->{last_line}))[0,1,2,3,4,5,9,10];
|
|
|
1365
|
|
|
1366 ### If a sequence has no reported alignments there will be a single output line with a bit-wise flag value of 4. We can store the next alignment and move on to the next Bowtie 2 instance
|
|
|
1367 if ($flag == 4){
|
|
|
1368 ## reading in the next alignment, which must be the next sequence
|
|
|
1369 my $newline = $fhs[$index]->{fh}-> getline();
|
|
|
1370 if ($newline){
|
|
|
1371 chomp $newline;
|
|
|
1372 my ($seq_id) = split (/\t/,$newline);
|
|
|
1373 $fhs[$index]->{last_seq_id} = $seq_id;
|
|
|
1374 $fhs[$index]->{last_line} = $newline;
|
|
|
1375 if ($seq_id eq $identifier){
|
|
|
1376 die "Sequence with ID $identifier did not produce any alignment, but next seq-ID was also $fhs[$index]->{last_seq_id}!\n";
|
|
|
1377 }
|
|
|
1378 next; # next instance
|
|
|
1379 }
|
|
|
1380 else{
|
|
|
1381 # assigning undef to last_seq_id and last_line and jumping to the next index (end of Bowtie 2 output)
|
|
|
1382 $fhs[$index]->{last_seq_id} = undef;
|
|
|
1383 $fhs[$index]->{last_line} = undef;
|
|
|
1384 next;
|
|
|
1385 }
|
|
|
1386 }
|
|
|
1387
|
|
|
1388 # if there are one or more proper alignments we can extract the chromosome number
|
|
|
1389 my $chromosome;
|
|
|
1390 if ($mapped_chromosome =~ s/_(CT|GA)_converted$//){
|
|
|
1391 $chromosome = $mapped_chromosome;
|
|
|
1392 }
|
|
|
1393 else{
|
|
|
1394 die "Chromosome number extraction failed for $mapped_chromosome\n";
|
|
|
1395 }
|
|
|
1396
|
|
|
1397 ### We will use the optional field to determine the best alignment. Later on we extract the number of mismatches and/or indels from the CIGAR string
|
|
|
1398 my ($alignment_score,$second_best,$MD_tag);
|
|
|
1399 my @fields = split (/\t/,$fhs[$index]->{last_line});
|
|
|
1400
|
|
|
1401 foreach (11..$#fields){
|
|
|
1402 if ($fields[$_] =~ /AS:i:(.*)/){
|
|
|
1403 $alignment_score = $1;
|
|
|
1404 }
|
|
|
1405 elsif ($fields[$_] =~ /XS:i:(.*)/){
|
|
|
1406 $second_best = $1;
|
|
|
1407 }
|
|
|
1408 elsif ($fields[$_] =~ /MD:Z:(.*)/){
|
|
|
1409 $MD_tag = $1;
|
|
|
1410 }
|
|
|
1411 }
|
|
|
1412
|
|
|
1413 # warn "First best alignment_score is: '$alignment_score'\n";
|
|
|
1414 # warn "MD tag is: '$MD_tag'\n";
|
|
|
1415 die "Failed to extract alignment score ($alignment_score) and MD tag ($MD_tag)!\n" unless (defined $alignment_score and defined $MD_tag);
|
|
|
1416
|
|
|
1417 if (defined $second_best){
|
|
|
1418 # warn "second best alignment_score is: '$second_best'\n";
|
|
|
1419
|
|
|
1420 # 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
|
|
|
1421 if ($alignment_score == $second_best){
|
|
|
1422 $alignment_ambiguous = 1;
|
|
|
1423 ## need to read and discard all additional ambiguous reads until we reach the next sequence
|
|
|
1424 until ($fhs[$index]->{last_seq_id} ne $identifier){
|
|
|
1425 my $newline = $fhs[$index]->{fh}-> getline();
|
|
|
1426 if ($newline){
|
|
|
1427 chomp $newline;
|
|
|
1428 my ($seq_id) = split (/\t/,$newline);
|
|
|
1429 $fhs[$index]->{last_seq_id} = $seq_id;
|
|
|
1430 $fhs[$index]->{last_line} = $newline;
|
|
|
1431 }
|
|
|
1432 else{
|
|
|
1433 # assigning undef to last_seq_id and last_line and jumping to the next index (end of Bowtie 2 output)
|
|
|
1434 $fhs[$index]->{last_seq_id} = undef;
|
|
|
1435 $fhs[$index]->{last_line} = undef;
|
|
|
1436 last; # break free in case we have reached the end of the alignment output
|
|
|
1437 }
|
|
|
1438 }
|
|
|
1439 # 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";
|
|
|
1440 }
|
|
|
1441 else{ # the next best alignment has a lower alignment score than the current read, so we can safely store the current alignment
|
|
|
1442
|
|
|
1443 my $alignment_location = join (":",$chromosome,$position);
|
|
|
1444
|
|
|
1445 ### 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
|
|
|
1446 ### 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
|
|
|
1447 ### 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
|
|
|
1448 ### 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
|
|
|
1449
|
|
|
1450 unless (exists $alignments{$alignment_location}){
|
|
|
1451 $alignments{$alignment_location}->{seq_id} = $id;
|
|
|
1452 $alignments{$alignment_location}->{alignment_score} = $alignment_score;
|
|
|
1453 $alignments{$alignment_location}->{bowtie_sequence} = $bowtie_sequence;
|
|
|
1454 $alignments{$alignment_location}->{index} = $index;
|
|
|
1455 $alignments{$alignment_location}->{chromosome} = $chromosome;
|
|
|
1456 $alignments{$alignment_location}->{position} = $position;
|
|
|
1457 $alignments{$alignment_location}->{CIGAR} = $cigar;
|
|
|
1458 $alignments{$alignment_location}->{MD_tag} = $MD_tag;
|
|
|
1459 }
|
|
|
1460
|
|
|
1461 ### now reading and discarding all (inferior) alignments of this sequencing read until we hit the next sequence
|
|
|
1462 until ($fhs[$index]->{last_seq_id} ne $identifier){
|
|
|
1463 my $newline = $fhs[$index]->{fh}-> getline();
|
|
|
1464 if ($newline){
|
|
|
1465 chomp $newline;
|
|
|
1466 my ($seq_id) = split (/\t/,$newline);
|
|
|
1467 $fhs[$index]->{last_seq_id} = $seq_id;
|
|
|
1468 $fhs[$index]->{last_line} = $newline;
|
|
|
1469 }
|
|
|
1470 else{
|
|
|
1471 # assigning undef to last_seq_id and last_line and jumping to the next index (end of Bowtie 2 output)
|
|
|
1472 $fhs[$index]->{last_seq_id} = undef;
|
|
|
1473 $fhs[$index]->{last_line} = undef;
|
|
|
1474 last; # break free in case we have reached the end of the alignment output
|
|
|
1475 }
|
|
|
1476 }
|
|
|
1477 # 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";
|
|
|
1478 }
|
|
|
1479 }
|
|
|
1480 else{ # there is no second best hit, so we can just store this one and read in the next sequence
|
|
|
1481
|
|
|
1482 my $alignment_location = join (":",$chromosome,$position);
|
|
|
1483
|
|
|
1484 ### 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
|
|
|
1485 ### 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
|
|
|
1486 ### 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
|
|
|
1487 ### 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
|
|
|
1488
|
|
|
1489 unless (exists $alignments{$alignment_location}){
|
|
|
1490 $alignments{$alignment_location}->{seq_id} = $id;
|
|
|
1491 $alignments{$alignment_location}->{alignment_score} = $alignment_score;
|
|
|
1492 $alignments{$alignment_location}->{bowtie_sequence} = $bowtie_sequence;
|
|
|
1493 $alignments{$alignment_location}->{index} = $index;
|
|
|
1494 $alignments{$alignment_location}->{chromosome} = $chromosome;
|
|
|
1495 $alignments{$alignment_location}->{position} = $position;
|
|
|
1496 $alignments{$alignment_location}->{MD_tag} = $MD_tag;
|
|
|
1497 $alignments{$alignment_location}->{CIGAR} = $cigar;
|
|
|
1498 }
|
|
|
1499
|
|
|
1500 my $newline = $fhs[$index]->{fh}-> getline();
|
|
|
1501 if ($newline){
|
|
|
1502 chomp $newline;
|
|
|
1503 my ($seq_id) = split (/\t/,$newline);
|
|
|
1504 $fhs[$index]->{last_seq_id} = $seq_id;
|
|
|
1505 $fhs[$index]->{last_line} = $newline;
|
|
|
1506 if ($seq_id eq $identifier){
|
|
|
1507 die "Sequence with ID $identifier did not have a second best alignment, but next seq-ID was also $fhs[$index]->{last_seq_id}!\n";
|
|
|
1508 }
|
|
|
1509 }
|
|
|
1510 else{
|
|
|
1511 # assigning undef to last_seq_id and last_line and jumping to the next index (end of Bowtie 2 output)
|
|
|
1512 $fhs[$index]->{last_seq_id} = undef;
|
|
|
1513 $fhs[$index]->{last_line} = undef;
|
|
|
1514 }
|
|
|
1515 }
|
|
|
1516 }
|
|
|
1517 }
|
|
|
1518
|
|
|
1519 ### 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.
|
|
|
1520 if ($alignment_ambiguous == 1){
|
|
|
1521 $counting{unsuitable_sequence_count}++;
|
|
|
1522 ### 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
|
|
|
1523 # my $ambiguous_read_output = join("\t",$identifier,'256','*','0','0','*','*','0','0',$sequence,$quality_value);
|
|
|
1524 # print "$ambiguous_read_output\n";
|
|
|
1525
|
|
|
1526 if ($ambiguous){
|
|
|
1527 return 2; # => exits to next sequence, and prints it out to _ambiguous_reads.txt if '--ambiguous' was specified
|
|
|
1528 }
|
|
|
1529 elsif ($unmapped){
|
|
|
1530 return 1; # => exits to next sequence, and prints it out to _unmapped_reads.txt if '--unmapped' but not '--ambiguous' was specified
|
|
|
1531 }
|
|
|
1532 else{
|
|
|
1533 return 0;
|
|
|
1534 }
|
|
|
1535 }
|
|
|
1536
|
|
|
1537 ### if there was no alignment found for a certain sequence at all we continue with the next sequence in the sequence file
|
|
|
1538 unless(%alignments){
|
|
|
1539 $counting{no_single_alignment_found}++;
|
|
|
1540 # my $unmapped_read_output = join("\t",$identifier,'4','*','0','0','*','*','0','0',$sequence,$quality_value);
|
|
|
1541 # print "$unmapped_read_output\n";
|
|
|
1542 if ($unmapped){
|
|
|
1543 return 1; # => exits to next sequence, and prints it out to _unmapped_reads.txt if '--unmapped' was specified
|
|
|
1544 }
|
|
|
1545 else{
|
|
|
1546 return 0; # default
|
|
|
1547 }
|
|
|
1548 }
|
|
|
1549
|
|
|
1550 #######################################################################################################################################################
|
|
|
1551
|
|
|
1552 ### 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
|
|
|
1553 ### 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)
|
|
|
1554 ### alignment score we are discarding the sequence altogether.
|
|
|
1555 ### 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
|
|
|
1556 ### opening (5) and extending (3 per bp) the gap.
|
|
|
1557
|
|
|
1558 #######################################################################################################################################################
|
|
|
1559
|
|
|
1560 my $methylation_call_params; # hash reference which will store all information we need for the methylation call
|
|
|
1561 my $sequence_fails = 0; # Going to use $sequence_fails as a 'memory' if a sequence could not be aligned uniquely (set to 1 then)
|
|
|
1562
|
|
|
1563 ### print contents of %alignments for debugging
|
|
|
1564 # if (scalar keys %alignments > 1){
|
|
|
1565 # print "\n******\n";
|
|
|
1566 # foreach my $alignment_location (sort {$a cmp $b} keys %alignments){
|
|
|
1567 # print "Loc: $alignment_location\n";
|
|
|
1568 # print "ID: $alignments{$alignment_location}->{seq_id}\n";
|
|
|
1569 # print "AS: $alignments{$alignment_location}->{alignment_score}\n";
|
|
|
1570 # print "Seq: $alignments{$alignment_location}->{bowtie_sequence}\n";
|
|
|
1571 # print "Index $alignments{$alignment_location}->{index}\n";
|
|
|
1572 # print "Chr: $alignments{$alignment_location}->{chromosome}\n";
|
|
|
1573 # print "pos: $alignments{$alignment_location}->{position}\n";
|
|
|
1574 # print "MD: $alignments{$alignment_location}->{MD_tag}\n\n";
|
|
|
1575 # }
|
|
|
1576 # print "\n******\n";
|
|
|
1577 # }
|
|
|
1578
|
|
|
1579 ### if there is only 1 entry in the hash with we accept it as the best alignment
|
|
|
1580 if (scalar keys %alignments == 1){
|
|
|
1581 for my $unique_best_alignment (keys %alignments){
|
|
|
1582 $methylation_call_params->{$identifier}->{bowtie_sequence} = $alignments{$unique_best_alignment}->{bowtie_sequence};
|
|
|
1583 $methylation_call_params->{$identifier}->{chromosome} = $alignments{$unique_best_alignment}->{chromosome};
|
|
|
1584 $methylation_call_params->{$identifier}->{position} = $alignments{$unique_best_alignment}->{position};
|
|
|
1585 $methylation_call_params->{$identifier}->{index} = $alignments{$unique_best_alignment}->{index};
|
|
|
1586 $methylation_call_params->{$identifier}->{alignment_score} = $alignments{$unique_best_alignment}->{alignment_score};
|
|
|
1587 $methylation_call_params->{$identifier}->{MD_tag} = $alignments{$unique_best_alignment}->{MD_tag};
|
|
|
1588 $methylation_call_params->{$identifier}->{CIGAR} = $alignments{$unique_best_alignment}->{CIGAR};
|
|
|
1589 }
|
|
|
1590 }
|
|
|
1591
|
|
|
1592 ### 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
|
|
|
1593 ### we boot the sequence altogether
|
|
|
1594 elsif (scalar keys %alignments >= 2 and scalar keys %alignments <= 4){
|
|
|
1595 my $best_alignment_score;
|
|
|
1596 my $best_alignment_location;
|
|
|
1597 foreach my $alignment_location (sort {$alignments{$b}->{alignment_score} <=> $alignments{$a}->{alignment_score}} keys %alignments){
|
|
|
1598 # print "$alignments{$alignment_location}->{alignment_score}\n";
|
|
|
1599 unless (defined $best_alignment_score){
|
|
|
1600 $best_alignment_score = $alignments{$alignment_location}->{alignment_score};
|
|
|
1601 $best_alignment_location = $alignment_location;
|
|
|
1602 # print "setting best alignment score: $best_alignment_score\n";
|
|
|
1603 }
|
|
|
1604 else{
|
|
|
1605 ### if the second best alignment has the same alignment score as the first one, the sequence will get booted
|
|
|
1606 if ($alignments{$alignment_location}->{alignment_score} == $best_alignment_score){
|
|
|
1607 # warn "Same alignment score, the sequence will get booted!\n";
|
|
|
1608 $sequence_fails = 1;
|
|
|
1609 last; # exiting after the second alignment since we know that the sequence has ambiguous alignments
|
|
|
1610 }
|
|
|
1611 ### else we are going to store the best alignment for further processing
|
|
|
1612 else{
|
|
|
1613 $methylation_call_params->{$identifier}->{bowtie_sequence} = $alignments{$best_alignment_location}->{bowtie_sequence};
|
|
|
1614 $methylation_call_params->{$identifier}->{chromosome} = $alignments{$best_alignment_location}->{chromosome};
|
|
|
1615 $methylation_call_params->{$identifier}->{position} = $alignments{$best_alignment_location}->{position};
|
|
|
1616 $methylation_call_params->{$identifier}->{index} = $alignments{$best_alignment_location}->{index};
|
|
|
1617 $methylation_call_params->{$identifier}->{alignment_score} = $alignments{$best_alignment_location}->{alignment_score};
|
|
|
1618 $methylation_call_params->{$identifier}->{MD_tag} = $alignments{$best_alignment_location}->{MD_tag};
|
|
|
1619 $methylation_call_params->{$identifier}->{CIGAR} = $alignments{$best_alignment_location}->{CIGAR};
|
|
|
1620 last; # exiting after processing the second alignment since the sequence produced a unique best alignment
|
|
|
1621 }
|
|
|
1622 }
|
|
|
1623 }
|
|
|
1624 }
|
|
|
1625 else{
|
|
|
1626 die "There are too many potential hits for this sequence (1-4 expected, but found: ",scalar keys %alignments,")\n";;
|
|
|
1627 }
|
|
|
1628
|
|
|
1629 ### skipping the sequence completely if there were multiple alignments with the same best alignment score at different positions
|
|
|
1630 if ($sequence_fails == 1){
|
|
|
1631 $counting{unsuitable_sequence_count}++;
|
|
|
1632
|
|
|
1633 ### 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
|
|
|
1634 # my $ambiguous_read_output = join("\t",$identifier,'256','*','0','0','*','*','0','0',$sequence,$quality_value);
|
|
|
1635 # print OUT "$ambiguous_read_output\n";
|
|
|
1636
|
|
|
1637 if ($ambiguous){
|
|
|
1638 return 2; # => exits to next sequence, and prints it out (in FastQ format) to _ambiguous_reads.txt if '--ambiguous' was specified
|
|
|
1639 }
|
|
|
1640 elsif ($unmapped){
|
|
|
1641 return 1; # => exits to next sequence, and prints it out (in FastQ format) to _unmapped_reads.txt if '--unmapped' but not '--ambiguous' was specified
|
|
|
1642 }
|
|
|
1643 else{
|
|
|
1644 return 0; # => exits to next sequence (default)
|
|
|
1645 }
|
|
|
1646 }
|
|
|
1647
|
|
|
1648 ### --DIRECTIONAL
|
|
|
1649 ### If the option --directional has been specified the user wants to consider only alignments to the original top strand or the original bottom strand. We will therefore
|
|
|
1650 ### discard all alignments to strands complementary to the original strands, as they should not exist in reality due to the library preparation protocol
|
|
|
1651 if ($directional){
|
|
|
1652 if ( ($methylation_call_params->{$identifier}->{index} == 2) or ($methylation_call_params->{$identifier}->{index} == 3) ){
|
|
|
1653 # warn "Alignment rejected! (index was: $methylation_call_params->{$identifier}->{index})\n";
|
|
|
1654 $counting{alignments_rejected_count}++;
|
|
|
1655 return 0;
|
|
|
1656 }
|
|
|
1657 }
|
|
|
1658
|
|
|
1659 ### If the sequence has not been rejected so far it has a unique best alignment
|
|
|
1660 $counting{unique_best_alignment_count}++;
|
|
|
1661
|
|
|
1662 ### Now we need to extract a genomic sequence that exactly corresponds to the reported alignment. This potentially means that we need to deal with insertions or deletions as well
|
|
|
1663 extract_corresponding_genomic_sequence_single_end_bowtie2 ($identifier,$methylation_call_params);
|
|
|
1664
|
|
|
1665 ### check test to see if the genomic sequence we extracted has the same length as the observed sequence+2, and only then we perform the methylation call
|
|
|
1666 if (length($methylation_call_params->{$identifier}->{unmodified_genomic_sequence}) != length($sequence)+2){
|
|
|
1667 warn "Chromosomal sequence could not be extracted for\t$identifier\t$methylation_call_params->{$identifier}->{chromosome}\t$methylation_call_params->{$identifier}->{position}\n";
|
|
|
1668 $counting{genomic_sequence_could_not_be_extracted_count}++;
|
|
|
1669 return 0;
|
|
|
1670 }
|
|
|
1671
|
|
|
1672
|
|
|
1673 ### otherwise we are set to perform the actual methylation call
|
|
|
1674 $methylation_call_params->{$identifier}->{methylation_call} = methylation_call($identifier,$sequence,$methylation_call_params->{$identifier}->{unmodified_genomic_sequence},$methylation_call_params->{$identifier}->{read_conversion});
|
|
|
1675 print_bisulfite_mapping_result_single_end_bowtie2 ($identifier,$sequence,$methylation_call_params,$quality_value);
|
|
|
1676 return 0; ## if a sequence got this far we do not want to print it to unmapped or ambiguous.out
|
|
|
1677 }
|
|
|
1678
|
|
|
1679
|
|
|
1680 sub determine_number_of_transliterations_performed{
|
|
|
1681 my ($sequence,$read_conversion) = @_;
|
|
|
1682 my $number_of_transliterations;
|
|
|
1683 if ($read_conversion eq 'CT'){
|
|
|
1684 $number_of_transliterations = $sequence =~ tr/C/T/;
|
|
|
1685 }
|
|
|
1686 elsif ($read_conversion eq 'GA'){
|
|
|
1687 $number_of_transliterations = $sequence =~ tr/G/A/;
|
|
|
1688 }
|
|
|
1689 else{
|
|
|
1690 die "Read conversion mode of the read was not specified $!\n";
|
|
|
1691 }
|
|
|
1692 return $number_of_transliterations;
|
|
|
1693 }
|
|
|
1694
|
|
|
1695 sub decide_whether_single_end_alignment_is_valid{
|
|
|
1696 my ($index,$identifier) = @_;
|
|
|
1697
|
|
|
1698 # extracting from Bowtie 1 format
|
|
|
1699 my ($id,$strand) = (split (/\t/,$fhs[$index]->{last_line}))[0,1];
|
|
|
1700
|
|
|
1701 ### ensuring that the entry is the correct sequence
|
|
|
1702 if (($id eq $fhs[$index]->{last_seq_id}) and ($id eq $identifier)){
|
|
|
1703 ### checking the orientation of the alignment. We need to discriminate between 8 different conditions, however only 4 of them are theoretically
|
|
|
1704 ### sensible alignments
|
|
|
1705 my $orientation = ensure_sensical_alignment_orientation_single_end ($index,$strand);
|
|
|
1706 ### If the orientation was correct can we move on
|
|
|
1707 if ($orientation == 1){
|
|
|
1708 return 1; ### 1st possibility for a sequence to pass
|
|
|
1709 }
|
|
|
1710 ### If the alignment was in the wrong orientation we need to read in a new line
|
|
|
1711 elsif($orientation == 0){
|
|
|
1712 my $newline = $fhs[$index]->{fh}->getline();
|
|
|
1713 if ($newline){
|
|
|
1714 ($id,$strand) = (split (/\t/,$newline))[0,1];
|
|
|
1715
|
|
|
1716 ### ensuring that the next entry is still the correct sequence
|
|
|
1717 if ($id eq $identifier){
|
|
|
1718 ### checking orientation again
|
|
|
1719 $orientation = ensure_sensical_alignment_orientation_single_end ($index,$strand);
|
|
|
1720 ### If the orientation was correct can we move on
|
|
|
1721 if ($orientation == 1){
|
|
|
1722 $fhs[$index]->{last_seq_id} = $id;
|
|
|
1723 $fhs[$index]->{last_line} = $newline;
|
|
|
1724 return 1; ### 2nd possibility for a sequence to pass
|
|
|
1725 }
|
|
|
1726 ### If the alignment was in the wrong orientation again we need to read in yet another new line and store it in @fhs
|
|
|
1727 elsif ($orientation == 0){
|
|
|
1728 $newline = $fhs[$index]->{fh}->getline();
|
|
|
1729 if ($newline){
|
|
|
1730 my ($seq_id) = split (/\t/,$newline);
|
|
|
1731 ### 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
|
|
|
1732 ### the same fields of the just read next entry
|
|
|
1733 die "Same seq ID 3 or more times in a row!(should be 2 max) $!" if ($seq_id eq $identifier);
|
|
|
1734 $fhs[$index]->{last_seq_id} = $seq_id;
|
|
|
1735 $fhs[$index]->{last_line} = $newline;
|
|
|
1736 return 0; # not processing anything this round as the alignment currently stored in last_line was in the wrong orientation
|
|
|
1737 }
|
|
|
1738 else{
|
|
|
1739 # assigning undef to last_seq_id and last_line (end of bowtie output)
|
|
|
1740 $fhs[$index]->{last_seq_id} = undef;
|
|
|
1741 $fhs[$index]->{last_line} = undef;
|
|
|
1742 return 0; # not processing anything as the alignment currently stored in last_line was in the wrong orientation
|
|
|
1743 }
|
|
|
1744 }
|
|
|
1745 else{
|
|
|
1746 die "The orientation of the alignment must be either correct or incorrect\n";
|
|
|
1747 }
|
|
|
1748 }
|
|
|
1749 ### the sequence we just read in is already the next sequence to be analysed -> store it in @fhs
|
|
|
1750 else{
|
|
|
1751 $fhs[$index]->{last_seq_id} = $id;
|
|
|
1752 $fhs[$index]->{last_line} = $newline;
|
|
|
1753 return 0; # processing the new alignment result only in the next round
|
|
|
1754 }
|
|
|
1755 }
|
|
|
1756 else {
|
|
|
1757 # assigning undef to last_seq_id and last_line (end of bowtie output)
|
|
|
1758 $fhs[$index]->{last_seq_id} = undef;
|
|
|
1759 $fhs[$index]->{last_line} = undef;
|
|
|
1760 return 0; # not processing anything as the alignment currently stored in last_line was in the wrong orientation
|
|
|
1761 }
|
|
|
1762 }
|
|
|
1763 else{
|
|
|
1764 die "The orientation of the alignment must be either correct or incorrect\n";
|
|
|
1765 }
|
|
|
1766 }
|
|
|
1767 ### the sequence stored in @fhs as last_line is already the next sequence to be analysed -> analyse next round
|
|
|
1768 else{
|
|
|
1769 return 0;
|
|
|
1770 }
|
|
|
1771 }
|
|
|
1772 #########################
|
|
|
1773 ### BOWTIE 1 | PAIRED-END
|
|
|
1774 #########################
|
|
|
1775
|
|
|
1776 sub check_bowtie_results_paired_ends{
|
|
|
1777 my ($sequence_1,$sequence_2,$identifier,$quality_value_1,$quality_value_2) = @_;
|
|
|
1778
|
|
|
1779 ### quality values are not given for FastA files, so they are initialised with a Phred quality of 40
|
|
|
1780 unless ($quality_value_1){
|
|
|
1781 $quality_value_1 = 'I'x(length$sequence_1);
|
|
|
1782 }
|
|
|
1783 unless ($quality_value_2){
|
|
|
1784 $quality_value_2 = 'I'x(length$sequence_2);
|
|
|
1785 }
|
|
|
1786
|
|
|
1787 # 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";
|
|
|
1788
|
|
|
1789 my %mismatches = ();
|
|
|
1790 ### reading from the bowtie output files to see if this sequence pair aligned to a bisulfite converted genome
|
|
|
1791
|
|
|
1792
|
|
|
1793 ### 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.
|
|
|
1794 ### alignments to the complementary strands are reported afterwards (CTOT got index 1, and CTOB got index 2).
|
|
|
1795 ### 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)
|
|
|
1796 ### 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
|
|
|
1797 ### strands are not being reported by specifying --directional
|
|
|
1798
|
|
|
1799 foreach my $index (0,3,1,2){
|
|
|
1800 ### skipping this index if the last alignment has been set to undefined already (i.e. end of bowtie output)
|
|
|
1801 next unless ($fhs[$index]->{last_line_1} and $fhs[$index]->{last_line_2} and defined $fhs[$index]->{last_seq_id});
|
|
|
1802 ### if the sequence pair we are currently looking at produced an alignment we are doing various things with it
|
|
|
1803 if ($fhs[$index]->{last_seq_id} eq $identifier) {
|
|
|
1804 # print "$identifier\n$fhs[$index]->{last_seq_id}\n\n";
|
|
|
1805
|
|
|
1806 ##################################################################################
|
|
|
1807 ### STEP I Processing the entry which is stored in last_line_1 and last_line_2 ###
|
|
|
1808 ##################################################################################
|
|
|
1809 my $valid_alignment_found = decide_whether_paired_end_alignment_is_valid($index,$identifier);
|
|
|
1810 ### sequences can fail at this point if there was only 1 alignment in the wrong orientation, or if there were 2 aligments both in the wrong
|
|
|
1811 ### orientation. We only continue to extract useful information about this alignment if 1 was returned
|
|
|
1812 if ($valid_alignment_found == 1){
|
|
|
1813 ### Bowtie outputs which made it this far are in the correct orientation, so we can continue to analyse the alignment itself.
|
|
|
1814 ### we store the useful information in %mismatches
|
|
|
1815 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];
|
|
|
1816 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];
|
|
|
1817 chomp $mismatch_info_1;
|
|
|
1818 chomp $mismatch_info_2;
|
|
|
1819
|
|
|
1820 ### need to extract the chromosome number from the bowtie output (which is either XY_CT_converted or XY_GA_converted
|
|
|
1821 my ($chromosome_1,$chromosome_2);
|
|
|
1822 if ($mapped_chromosome_1 =~ s/_(CT|GA)_converted$//){
|
|
|
1823 $chromosome_1 = $mapped_chromosome_1;
|
|
|
1824 }
|
|
|
1825 else{
|
|
|
1826 die "Chromosome number extraction failed for $mapped_chromosome_1\n";
|
|
|
1827 }
|
|
|
1828 if ($mapped_chromosome_2 =~ s/_(CT|GA)_converted$//){
|
|
|
1829 $chromosome_2 = $mapped_chromosome_2;
|
|
|
1830 }
|
|
|
1831 else{
|
|
|
1832 die "Chromosome number extraction failed for $mapped_chromosome_2\n";
|
|
|
1833 }
|
|
|
1834
|
|
|
1835 ### Now extracting the number of mismatches to the converted genome
|
|
|
1836 my $number_of_mismatches_1;
|
|
|
1837 my $number_of_mismatches_2;
|
|
|
1838 if ($mismatch_info_1 eq ''){
|
|
|
1839 $number_of_mismatches_1 = 0;
|
|
|
1840 }
|
|
|
1841 elsif ($mismatch_info_1 =~ /^\d/){
|
|
|
1842 my @mismatches = split (/,/,$mismatch_info_1);
|
|
|
1843 $number_of_mismatches_1 = scalar @mismatches;
|
|
|
1844 }
|
|
|
1845 else{
|
|
|
1846 die "Something weird is going on with the mismatch field\n";
|
|
|
1847 }
|
|
|
1848 if ($mismatch_info_2 eq ''){
|
|
|
1849 $number_of_mismatches_2 = 0;
|
|
|
1850 }
|
|
|
1851 elsif ($mismatch_info_2 =~ /^\d/){
|
|
|
1852 my @mismatches = split (/,/,$mismatch_info_2);
|
|
|
1853 $number_of_mismatches_2 = scalar @mismatches;
|
|
|
1854 }
|
|
|
1855 else{
|
|
|
1856 die "Something weird is going on with the mismatch field\n";
|
|
|
1857 }
|
|
|
1858 ### To decide whether a sequence pair has a unique best alignment we will look at the lowest sum of mismatches from both alignments
|
|
|
1859 my $sum_of_mismatches = $number_of_mismatches_1+$number_of_mismatches_2;
|
|
|
1860 ### creating a composite location variable from $chromosome and $position and storing the alignment information in a temporary hash table
|
|
|
1861 die "Position 1 is higher than position 2" if ($position_1 > $position_2);
|
|
|
1862 die "Paired-end alignments need to be on the same chromosome\n" unless ($chromosome_1 eq $chromosome_2);
|
|
|
1863 my $alignment_location = join(":",$chromosome_1,$position_1,$position_2);
|
|
|
1864 ### 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
|
|
|
1865 ### strand) were methylated and therefore protected. It is not needed to overwrite the same positional entry with a second entry for the same
|
|
|
1866 ### location (the genomic sequence extraction and methylation would not be affected by this, only the thing which would change is the index
|
|
|
1867 ### number for the found alignment)
|
|
|
1868 unless (exists $mismatches{$sum_of_mismatches}->{$alignment_location}){
|
|
|
1869 $mismatches{$sum_of_mismatches}->{$alignment_location}->{seq_id}=$id_1; # either is fine
|
|
|
1870 $mismatches{$sum_of_mismatches}->{$alignment_location}->{bowtie_sequence_1}=$bowtie_sequence_1;
|
|
|
1871 $mismatches{$sum_of_mismatches}->{$alignment_location}->{bowtie_sequence_2}=$bowtie_sequence_2;
|
|
|
1872 $mismatches{$sum_of_mismatches}->{$alignment_location}->{index}=$index;
|
|
|
1873 $mismatches{$sum_of_mismatches}->{$alignment_location}->{chromosome}=$chromosome_1; # either is fine
|
|
|
1874 $mismatches{$sum_of_mismatches}->{$alignment_location}->{start_seq_1}=$position_1;
|
|
|
1875 $mismatches{$sum_of_mismatches}->{$alignment_location}->{start_seq_2}=$position_2;
|
|
|
1876 $mismatches{$sum_of_mismatches}->{$alignment_location}->{number_of_mismatches_1} = $number_of_mismatches_1;
|
|
|
1877 $mismatches{$sum_of_mismatches}->{$alignment_location}->{number_of_mismatches_2} = $number_of_mismatches_2;
|
|
|
1878 }
|
|
|
1879 ###################################################################################################################################################
|
|
|
1880 ### 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 ###
|
|
|
1881 ### 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. ###
|
|
|
1882 ### 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 ###
|
|
|
1883 ### this round ###
|
|
|
1884 ###################################################################################################################################################
|
|
|
1885 my $newline_1 = $fhs[$index]->{fh}-> getline();
|
|
|
1886 my $newline_2 = $fhs[$index]->{fh}-> getline();
|
|
|
1887
|
|
|
1888 if ($newline_1 and $newline_2){
|
|
|
1889 my ($seq_id_1) = split (/\t/,$newline_1);
|
|
|
1890 my ($seq_id_2) = split (/\t/,$newline_2);
|
|
|
1891
|
|
|
1892 if ($seq_id_1 =~ s/\/1$//){ # removing the read /1 tag
|
|
|
1893 $fhs[$index]->{last_seq_id} = $seq_id_1;
|
|
|
1894 }
|
|
|
1895 elsif ($seq_id_2 =~ s/\/1$//){ # removing the read /1 tag
|
|
|
1896 $fhs[$index]->{last_seq_id} = $seq_id_2;
|
|
|
1897 }
|
|
|
1898 else{
|
|
|
1899 die "Either read 1 or read 2 needs to end on '/1'\n";
|
|
|
1900 }
|
|
|
1901
|
|
|
1902 $fhs[$index]->{last_line_1} = $newline_1;
|
|
|
1903 $fhs[$index]->{last_line_2} = $newline_2;
|
|
|
1904 }
|
|
|
1905 else {
|
|
|
1906 # assigning undef to last_seq_id and both last_lines and jumping to the next index (end of bowtie output)
|
|
|
1907 $fhs[$index]->{last_seq_id} = undef;
|
|
|
1908 $fhs[$index]->{last_line_1} = undef;
|
|
|
1909 $fhs[$index]->{last_line_2} = undef;
|
|
|
1910 next; # jumping to the next index
|
|
|
1911 }
|
|
|
1912 ### Now processing the entry we just stored in last_line_1 and last_line_2
|
|
|
1913 $valid_alignment_found = decide_whether_paired_end_alignment_is_valid($index,$identifier);
|
|
|
1914 ### only processing the alignment further if 1 was returned. 0 will be returned either if the alignment is already the next sequence pair to
|
|
|
1915 ### be analysed or if it was a second alignment of the current sequence pair but in the wrong orientation
|
|
|
1916 if ($valid_alignment_found == 1){
|
|
|
1917 ### we store the useful information in %mismatches
|
|
|
1918 ($id_1,$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];
|
|
|
1919 ($id_2,$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];
|
|
|
1920 chomp $mismatch_info_1;
|
|
|
1921 chomp $mismatch_info_2;
|
|
|
1922 ### need to extract the chromosome number from the bowtie output (which is either _CT_converted or _GA_converted)
|
|
|
1923 if ($mapped_chromosome_1 =~ s/_(CT|GA)_converted$//){
|
|
|
1924 $chromosome_1 = $mapped_chromosome_1;
|
|
|
1925 }
|
|
|
1926 else{
|
|
|
1927 die "Chromosome number extraction failed for $mapped_chromosome_1\n";
|
|
|
1928 }
|
|
|
1929 if ($mapped_chromosome_2 =~ s/_(CT|GA)_converted$//){
|
|
|
1930 $chromosome_2 = $mapped_chromosome_2;
|
|
|
1931 }
|
|
|
1932 else{
|
|
|
1933 die "Chromosome number extraction failed for $mapped_chromosome_2\n";
|
|
|
1934 }
|
|
|
1935
|
|
|
1936 $number_of_mismatches_1='';
|
|
|
1937 $number_of_mismatches_2='';
|
|
|
1938 ### Now extracting the number of mismatches to the converted genome
|
|
|
1939 if ($mismatch_info_1 eq ''){
|
|
|
1940 $number_of_mismatches_1 = 0;
|
|
|
1941 }
|
|
|
1942 elsif ($mismatch_info_1 =~ /^\d/){
|
|
|
1943 my @mismatches = split (/,/,$mismatch_info_1);
|
|
|
1944 $number_of_mismatches_1 = scalar @mismatches;
|
|
|
1945 }
|
|
|
1946 else{
|
|
|
1947 die "Something weird is going on with the mismatch field\n";
|
|
|
1948 }
|
|
|
1949 if ($mismatch_info_2 eq ''){
|
|
|
1950 $number_of_mismatches_2 = 0;
|
|
|
1951 }
|
|
|
1952 elsif ($mismatch_info_2 =~ /^\d/){
|
|
|
1953 my @mismatches = split (/,/,$mismatch_info_2);
|
|
|
1954 $number_of_mismatches_2 = scalar @mismatches;
|
|
|
1955 }
|
|
|
1956 else{
|
|
|
1957 die "Something weird is going on with the mismatch field\n";
|
|
|
1958 }
|
|
|
1959 ### To decide whether a sequence pair has a unique best alignment we will look at the lowest sum of mismatches from both alignments
|
|
|
1960 $sum_of_mismatches = $number_of_mismatches_1+$number_of_mismatches_2;
|
|
|
1961 ### creating a composite location variable from $chromosome and $position and storing the alignment information in a temporary hash table
|
|
|
1962 die "position 1 is greater than position 2" if ($position_1 > $position_2);
|
|
|
1963 die "Paired-end alignments need to be on the same chromosome\n" unless ($chromosome_1 eq $chromosome_2);
|
|
|
1964 $alignment_location = join(":",$chromosome_1,$position_1,$position_2);
|
|
|
1965 ### 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
|
|
|
1966 ### strand) were methylated and therefore protected. It is not needed to overwrite the same positional entry with a second entry for the same
|
|
|
1967 ### location (the genomic sequence extraction and methylation would not be affected by this, only the thing which would change is the index
|
|
|
1968 ### number for the found alignment)
|
|
|
1969 unless (exists $mismatches{$sum_of_mismatches}->{$alignment_location}){
|
|
|
1970 $mismatches{$sum_of_mismatches}->{$alignment_location}->{seq_id}=$id_1; # either is fine
|
|
|
1971 $mismatches{$sum_of_mismatches}->{$alignment_location}->{bowtie_sequence_1}=$bowtie_sequence_1;
|
|
|
1972 $mismatches{$sum_of_mismatches}->{$alignment_location}->{bowtie_sequence_2}=$bowtie_sequence_2;
|
|
|
1973 $mismatches{$sum_of_mismatches}->{$alignment_location}->{index}=$index;
|
|
|
1974 $mismatches{$sum_of_mismatches}->{$alignment_location}->{chromosome}=$chromosome_1; # either is fine
|
|
|
1975 $mismatches{$sum_of_mismatches}->{$alignment_location}->{start_seq_1}=$position_1;
|
|
|
1976 $mismatches{$sum_of_mismatches}->{$alignment_location}->{start_seq_2}=$position_2;
|
|
|
1977 $mismatches{$sum_of_mismatches}->{$alignment_location}->{number_of_mismatches_1} = $number_of_mismatches_1;
|
|
|
1978 $mismatches{$sum_of_mismatches}->{$alignment_location}->{number_of_mismatches_2} = $number_of_mismatches_2;
|
|
|
1979 }
|
|
|
1980 ###############################################################################################################################################
|
|
|
1981 ### 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 ###
|
|
|
1982 ###############################################################################################################################################
|
|
|
1983 $newline_1 = $fhs[$index]->{fh}-> getline();
|
|
|
1984 $newline_2 = $fhs[$index]->{fh}-> getline();
|
|
|
1985
|
|
|
1986 if ($newline_1 and $newline_2){
|
|
|
1987 my ($seq_id_1) = split (/\t/,$newline_1);
|
|
|
1988 my ($seq_id_2) = split (/\t/,$newline_2);
|
|
|
1989
|
|
|
1990 if ($seq_id_1 =~ s/\/1$//){ # removing the read /1 tag
|
|
|
1991 $fhs[$index]->{last_seq_id} = $seq_id_1;
|
|
|
1992 }
|
|
|
1993 if ($seq_id_2 =~ s/\/1$//){ # removing the read /1 tag
|
|
|
1994 $fhs[$index]->{last_seq_id} = $seq_id_2;
|
|
|
1995 }
|
|
|
1996 $fhs[$index]->{last_line_1} = $newline_1;
|
|
|
1997 $fhs[$index]->{last_line_2} = $newline_2;
|
|
|
1998 }
|
|
|
1999 else {
|
|
|
2000 # assigning undef to last_seq_id and both last_lines and jumping to the next index (end of bowtie output)
|
|
|
2001 $fhs[$index]->{last_seq_id} = undef;
|
|
|
2002 $fhs[$index]->{last_line_1} = undef;
|
|
|
2003 $fhs[$index]->{last_line_2} = undef;
|
|
|
2004 next; # jumping to the next index
|
|
|
2005 }
|
|
|
2006 ### within the 2nd sequence pair alignment in correct orientation found
|
|
|
2007 }
|
|
|
2008 ### within the 1st sequence pair alignment in correct orientation found
|
|
|
2009 }
|
|
|
2010 ### still within the (last_seq_id eq identifier) condition
|
|
|
2011 }
|
|
|
2012 ### still within foreach index loop
|
|
|
2013 }
|
|
|
2014 ### if there was no single alignment found for a certain sequence we will continue with the next sequence in the sequence file
|
|
|
2015 unless(%mismatches){
|
|
|
2016 $counting{no_single_alignment_found}++;
|
|
|
2017 return 1; ### We will print this sequence out as unmapped sequence if --un unmapped.out has been specified
|
|
|
2018 }
|
|
|
2019 ### Going to use the variable $sequence_pair_fails as a 'memory' if a sequence could not be aligned uniquely (set to 1 then)
|
|
|
2020 my $sequence_pair_fails = 0;
|
|
|
2021 ### Declaring an empty hash reference which will store all information we need for the methylation call
|
|
|
2022 my $methylation_call_params; # hash reference!
|
|
|
2023 ### 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
|
|
|
2024 ### 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
|
|
|
2025 ### meth_call variables, if there are multiple hits with the same amount of (lowest) mismatches we are discarding the sequence altogether
|
|
|
2026 foreach my $mismatch_number (sort {$a<=>$b} keys %mismatches){
|
|
|
2027 #dev print "Number of mismatches: $mismatch_number\t$identifier\t$sequence_1\t$sequence_2\n";
|
|
|
2028 foreach my $entry (keys (%{$mismatches{$mismatch_number}}) ){
|
|
|
2029 #dev print "$mismatch_number\t$entry\t$mismatches{$mismatch_number}->{$entry}->{index}\n";
|
|
|
2030 # 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}->{$entry}->{index}),"\n";
|
|
|
2031 }
|
|
|
2032 if (scalar keys %{$mismatches{$mismatch_number}} == 1){
|
|
|
2033 # print "Unique best alignment for sequence pair $sequence_1\t$sequence_1\n";
|
|
|
2034 for my $unique_best_alignment (keys %{$mismatches{$mismatch_number}}){
|
|
|
2035 $methylation_call_params->{$identifier}->{seq_id} = $identifier;
|
|
|
2036 $methylation_call_params->{$identifier}->{bowtie_sequence_1} = $mismatches{$mismatch_number}->{$unique_best_alignment}->{bowtie_sequence_1};
|
|
|
2037 $methylation_call_params->{$identifier}->{bowtie_sequence_2} = $mismatches{$mismatch_number}->{$unique_best_alignment}->{bowtie_sequence_2};
|
|
|
2038 $methylation_call_params->{$identifier}->{chromosome} = $mismatches{$mismatch_number}->{$unique_best_alignment}->{chromosome};
|
|
|
2039 $methylation_call_params->{$identifier}->{start_seq_1} = $mismatches{$mismatch_number}->{$unique_best_alignment}->{start_seq_1};
|
|
|
2040 $methylation_call_params->{$identifier}->{start_seq_2} = $mismatches{$mismatch_number}->{$unique_best_alignment}->{start_seq_2};
|
|
|
2041 $methylation_call_params->{$identifier}->{alignment_end} = ($mismatches{$mismatch_number}->{$unique_best_alignment}->{start_seq_2}+length($mismatches{$mismatch_number}->{$unique_best_alignment}->{bowtie_sequence_2}));
|
|
|
2042 $methylation_call_params->{$identifier}->{index} = $mismatches{$mismatch_number}->{$unique_best_alignment}->{index};
|
|
|
2043 $methylation_call_params->{$identifier}->{number_of_mismatches_1} = $mismatches{$mismatch_number}->{$unique_best_alignment}->{number_of_mismatches_1};
|
|
|
2044 $methylation_call_params->{$identifier}->{number_of_mismatches_2} = $mismatches{$mismatch_number}->{$unique_best_alignment}->{number_of_mismatches_2};
|
|
|
2045 }
|
|
|
2046 }
|
|
|
2047 else{
|
|
|
2048 $sequence_pair_fails = 1;
|
|
|
2049 }
|
|
|
2050 ### after processing the alignment with the lowest number of mismatches we exit
|
|
|
2051 last;
|
|
|
2052 }
|
|
|
2053 ### skipping the sequence completely if there were multiple alignments with the same amount of lowest mismatches found at different positions
|
|
|
2054 if ($sequence_pair_fails == 1){
|
|
|
2055 $counting{unsuitable_sequence_count}++;
|
|
|
2056 if ($ambiguous){
|
|
|
2057 return 2; # => exits to next sequence pair, and prints both seqs out to multiple_alignments_1 and -2 if --ambiguous has been specified
|
|
|
2058 }
|
|
|
2059 if ($unmapped){
|
|
|
2060 return 1; # => exits to next sequence pair, and prints both seqs out to unmapped_1 and _2 if --un has been specified
|
|
|
2061 }
|
|
|
2062 else{
|
|
|
2063 return 0; # => exits to next sequence (default)
|
|
|
2064 }
|
|
|
2065 }
|
|
|
2066
|
|
|
2067 ### --DIRECTIONAL
|
|
|
2068 ### If the option --directional has been specified the user wants to consider only alignments to the original top strand or the original bottom strand. We will therefore
|
|
|
2069 ### discard all alignments to strands complementary to the original strands, as they should not exist in reality due to the library preparation protocol
|
|
|
2070 if ($directional){
|
|
|
2071 if ( ($methylation_call_params->{$identifier}->{index} == 1) or ($methylation_call_params->{$identifier}->{index} == 2) ){
|
|
|
2072 # warn "Alignment rejected! (index was: $methylation_call_params->{$identifier}->{index})\n";
|
|
|
2073 $counting{alignments_rejected_count}++;
|
|
|
2074 return 0;
|
|
|
2075 }
|
|
|
2076 }
|
|
|
2077
|
|
|
2078 ### If the sequence has not been rejected so far it does have a unique best alignment
|
|
|
2079 $counting{unique_best_alignment_count}++;
|
|
|
2080 extract_corresponding_genomic_sequence_paired_ends($identifier,$methylation_call_params);
|
|
|
2081
|
|
|
2082 ### check test to see if the genomic sequences we extracted has the same length as the observed sequences +2, and only then we perform the methylation call
|
|
|
2083 if (length($methylation_call_params->{$identifier}->{unmodified_genomic_sequence_1}) != length($sequence_1)+2){
|
|
|
2084 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";
|
|
|
2085 $counting{genomic_sequence_could_not_be_extracted_count}++;
|
|
|
2086 return 0;
|
|
|
2087 }
|
|
|
2088 if (length($methylation_call_params->{$identifier}->{unmodified_genomic_sequence_2}) != length($sequence_2)+2){
|
|
|
2089 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";
|
|
|
2090 $counting{genomic_sequence_could_not_be_extracted_count}++;
|
|
|
2091 return 0;
|
|
|
2092 }
|
|
|
2093
|
|
|
2094 ### otherwise we are set to perform the actual methylation call
|
|
|
2095 $methylation_call_params->{$identifier}->{methylation_call_1} = methylation_call($identifier,$sequence_1,$methylation_call_params->{$identifier}->{unmodified_genomic_sequence_1},$methylation_call_params->{$identifier}->{read_conversion_1});
|
|
|
2096 $methylation_call_params->{$identifier}->{methylation_call_2} = methylation_call($identifier,$sequence_2,$methylation_call_params->{$identifier}->{unmodified_genomic_sequence_2},$methylation_call_params->{$identifier}->{read_conversion_2});
|
|
|
2097
|
|
|
2098 print_bisulfite_mapping_results_paired_ends($identifier,$sequence_1,$sequence_2,$methylation_call_params,$quality_value_1,$quality_value_2);
|
|
|
2099 return 0; ## otherwise 1 will be returned by default, which would print the sequence pair to unmapped_1 and _2
|
|
|
2100 }
|
|
|
2101
|
|
|
2102 #########################
|
|
|
2103 ### BOWTIE 2 | PAIRED-END
|
|
|
2104 #########################
|
|
|
2105
|
|
|
2106 sub check_bowtie_results_paired_ends_bowtie2{
|
|
|
2107 my ($sequence_1,$sequence_2,$identifier,$quality_value_1,$quality_value_2) = @_;
|
|
|
2108
|
|
|
2109 ### quality values are not given for FastA files, so they are initialised with a Phred quality of 40
|
|
|
2110 unless ($quality_value_1){
|
|
|
2111 $quality_value_1 = 'I'x(length$sequence_1);
|
|
|
2112 }
|
|
|
2113
|
|
|
2114 unless ($quality_value_2){
|
|
|
2115 $quality_value_2 = 'I'x(length$sequence_2);
|
|
|
2116 }
|
|
|
2117
|
|
|
2118
|
|
|
2119 # 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";
|
|
|
2120
|
|
|
2121
|
|
|
2122 my %alignments;
|
|
|
2123 my $alignment_ambiguous = 0;
|
|
|
2124
|
|
|
2125 ### reading from the Bowtie 2 output filehandles
|
|
|
2126
|
|
|
2127 ### 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.
|
|
|
2128 ### alignments to the complementary strands are reported afterwards (CTOT got index 1, and CTOB got index 2).
|
|
|
2129 ### 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)
|
|
|
2130 ### 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
|
|
|
2131 ### strands are not being reported when '--directional' is specified
|
|
|
2132
|
|
|
2133 foreach my $index (0,3,1,2){
|
|
|
2134 ### skipping this index if the last alignment has been set to undefined already (i.e. end of bowtie output)
|
|
|
2135 next unless ($fhs[$index]->{last_line_1} and $fhs[$index]->{last_line_2} and defined $fhs[$index]->{last_seq_id});
|
|
|
2136
|
|
|
2137 ### if the sequence pair we are currently looking at produced an alignment we are doing various things with it
|
|
|
2138 if ($fhs[$index]->{last_seq_id} eq $identifier) {
|
|
|
2139
|
|
|
2140 my ($id_1,$flag_1,$mapped_chromosome_1,$position_1,$mapping_quality_1,$cigar_1,$bowtie_sequence_1,$qual_1) = (split (/\t/,$fhs[$index]->{last_line_1}))[0,1,2,3,4,5,9,10];
|
|
|
2141 my ($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];
|
|
|
2142 # print "Index: $index\t$fhs[$index]->{last_line_1}\n";
|
|
|
2143 # print "Index: $index\t$fhs[$index]->{last_line_2}\n";
|
|
|
2144 # print join ("\t",$id_1,$flag_1,$mapped_chromosome_1,$position_1,$mapping_quality_1,$cigar_1,$bowtie_sequence_1,$qual_1),"\n";
|
|
|
2145 # print join ("\t",$id_2,$flag_2,$mapped_chromosome_2,$position_2,$mapping_quality_2,$cigar_2,$bowtie_sequence_2,$qual_2),"\n";
|
|
|
2146 $id_1 =~ s/\/1$//;
|
|
|
2147 $id_2 =~ s/\/2$//;
|
|
|
2148
|
|
|
2149 # SAM format specifications for Bowtie 2
|
|
|
2150 # (1) Name of read that aligned
|
|
|
2151 # (2) Sum of all applicable flags. Flags relevant to Bowtie are:
|
|
|
2152 # 1 The read is one of a pair
|
|
|
2153 # 2 The alignment is one end of a proper paired-end alignment
|
|
|
2154 # 4 The read has no reported alignments
|
|
|
2155 # 8 The read is one of a pair and has no reported alignments
|
|
|
2156 # 16 The alignment is to the reverse reference strand
|
|
|
2157 # 32 The other mate in the paired-end alignment is aligned to the reverse reference strand
|
|
|
2158 # 64 The read is mate 1 in a pair
|
|
|
2159 # 128 The read is mate 2 in a pair
|
|
|
2160 # 256 The read has multiple mapping states
|
|
|
2161 # (3) Name of reference sequence where alignment occurs (unmapped reads have a *)
|
|
|
2162 # (4) 1-based offset into the forward reference strand where leftmost character of the alignment occurs (0 for unmapped reads)
|
|
|
2163 # (5) Mapping quality (255 means MAPQ is not available)
|
|
|
2164 # (6) CIGAR string representation of alignment (* if unavailable)
|
|
|
2165 # (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.
|
|
|
2166 # (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.
|
|
|
2167 # (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.
|
|
|
2168 # (10) Read sequence (reverse-complemented if aligned to the reverse strand)
|
|
|
2169 # (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.
|
|
|
2170 # (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:
|
|
|
2171 # 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.
|
|
|
2172 # 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.
|
|
|
2173 # 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.
|
|
|
2174 # XN:i:<N> The number of ambiguous bases in the reference covering this alignment. Only present if SAM record is for an aligned read.
|
|
|
2175 # XM:i:<N> The number of mismatches in the alignment. Only present if SAM record is for an aligned read.
|
|
|
2176 # 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.
|
|
|
2177 # 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.
|
|
|
2178 # 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.
|
|
|
2179 # YF:Z:<N> String indicating reason why the read was filtered out. See also: Filtering. Only appears for reads that were filtered out.
|
|
|
2180 # 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.
|
|
|
2181
|
|
|
2182 ### 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).
|
|
|
2183 ### We can store the next alignment and move on to the next Bowtie 2 instance
|
|
|
2184 if ($flag_1 == 77 and $flag_2 == 141){
|
|
|
2185 ## reading in the next alignment, which must be the next sequence
|
|
|
2186 my $newline_1 = $fhs[$index]->{fh}-> getline();
|
|
|
2187 my $newline_2 = $fhs[$index]->{fh}-> getline();
|
|
|
2188
|
|
|
2189 if ($newline_1 and $newline_2){
|
|
|
2190 chomp $newline_1;
|
|
|
2191 chomp $newline_2;
|
|
|
2192 my ($seq_id_1) = split (/\t/,$newline_1);
|
|
|
2193 my ($seq_id_2) = split (/\t/,$newline_2);
|
|
|
2194 $seq_id_1 =~ s/\/1$//;
|
|
|
2195 $seq_id_2 =~ s/\/2$//;
|
|
|
2196 $fhs[$index]->{last_seq_id} = $seq_id_1;
|
|
|
2197 $fhs[$index]->{last_line_1} = $newline_1;
|
|
|
2198 $fhs[$index]->{last_line_2} = $newline_2;
|
|
|
2199
|
|
|
2200 # print "current sequence ($identifier) did not map, reading in next sequence\n";
|
|
|
2201 # print "$index\t$fhs[$index]->{last_seq_id}\n";
|
|
|
2202 # print "$index\t$fhs[$index]->{last_line_1}\n";
|
|
|
2203 # print "$index\t$fhs[$index]->{last_line_2}\n";
|
|
|
2204 next; # next instance
|
|
|
2205 }
|
|
|
2206 else{
|
|
|
2207 # assigning undef to last_seq_id and last_line and jumping to the next index (end of Bowtie 2 output)
|
|
|
2208 $fhs[$index]->{last_seq_id} = undef;
|
|
|
2209 $fhs[$index]->{last_line_1} = undef;
|
|
|
2210 $fhs[$index]->{last_line_2} = undef;
|
|
|
2211 next;
|
|
|
2212 }
|
|
|
2213 }
|
|
|
2214
|
|
|
2215 ### If there are one or more proper alignments we can extract the chromosome number
|
|
|
2216 my ($chromosome_1,$chromosome_2);
|
|
|
2217 if ($mapped_chromosome_1 =~ s/_(CT|GA)_converted$//){
|
|
|
2218 $chromosome_1 = $mapped_chromosome_1;
|
|
|
2219 }
|
|
|
2220 else{
|
|
|
2221 die "Chromosome number extraction failed for $mapped_chromosome_1\n";
|
|
|
2222 }
|
|
|
2223 if ($mapped_chromosome_2 =~ s/_(CT|GA)_converted$//){
|
|
|
2224 $chromosome_2 = $mapped_chromosome_2;
|
|
|
2225 }
|
|
|
2226 else{
|
|
|
2227 die "Chromosome number extraction failed for $mapped_chromosome_2\n";
|
|
|
2228 }
|
|
|
2229
|
|
|
2230 die "Paired-end alignments need to be on the same chromosome\n" unless ($chromosome_1 eq $chromosome_2);
|
|
|
2231
|
|
|
2232 ### We will use the optional fields to determine the best alignments. Later on we extract the number of mismatches and/or indels from the CIGAR string
|
|
|
2233 my ($alignment_score_1,$alignment_score_2,$second_best_1,$second_best_2,$MD_tag_1,$MD_tag_2);
|
|
|
2234
|
|
|
2235 my @fields_1 = split (/\t/,$fhs[$index]->{last_line_1});
|
|
|
2236 my @fields_2 = split (/\t/,$fhs[$index]->{last_line_2});
|
|
|
2237
|
|
|
2238 foreach (11..$#fields_1){
|
|
|
2239 if ($fields_1[$_] =~ /AS:i:(.*)/){
|
|
|
2240 $alignment_score_1 = $1;
|
|
|
2241 }
|
|
|
2242 elsif ($fields_1[$_] =~ /XS:i:(.*)/){
|
|
|
2243 $second_best_1 = $1;
|
|
|
2244 }
|
|
|
2245 elsif ($fields_1[$_] =~ /MD:Z:(.*)/){
|
|
|
2246 $MD_tag_1 = $1;
|
|
|
2247 }
|
|
|
2248 }
|
|
|
2249
|
|
|
2250 foreach (11..$#fields_2){
|
|
|
2251 if ($fields_2[$_] =~ /AS:i:(.*)/){
|
|
|
2252 $alignment_score_2 = $1;
|
|
|
2253 }
|
|
|
2254 elsif ($fields_2[$_] =~ /XS:i:(.*)/){
|
|
|
2255 $second_best_2 = $1;
|
|
|
2256 }
|
|
|
2257 elsif ($fields_2[$_] =~ /MD:Z:(.*)/){
|
|
|
2258 $MD_tag_2 = $1;
|
|
|
2259 }
|
|
|
2260 }
|
|
|
2261
|
|
|
2262 die "Failed to extract alignment score 1 ($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 $MD_tag_1);
|
|
|
2263 die "Failed to extract alignment score 2 ($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 $MD_tag_2);
|
|
|
2264
|
|
|
2265 # warn "First read 1 alignment score is: '$alignment_score_1'\n";
|
|
|
2266 # warn "First read 2 alignment score is: '$alignment_score_2'\n";
|
|
|
2267 # warn "MD tag 1 is: '$MD_tag_1'\n";
|
|
|
2268 # warn "MD tag 2 is: '$MD_tag_2'\n";
|
|
|
2269
|
|
|
2270 ### To decide whether a sequence pair has a unique best alignment we will look at the highest sum of alignment scores from both alignments
|
|
|
2271 my $sum_of_alignment_scores_1 = $alignment_score_1 + $alignment_score_2 ;
|
|
|
2272 # print "sum of alignment scores: $sum_of_alignment_scores_1\n\n";
|
|
|
2273
|
|
|
2274 if (defined $second_best_1 and defined $second_best_2){
|
|
|
2275 my $sum_of_alignment_scores_second_best = $second_best_1 + $second_best_2;
|
|
|
2276 # warn "Second best alignment_score_1 is: '$second_best_1'\n";
|
|
|
2277 # warn "Second best alignment_score_2 is: '$second_best_2'\n";
|
|
|
2278 # warn "Second best alignment sum of alignment scores is: '$sum_of_alignment_scores_second_best'\n";
|
|
|
2279
|
|
|
2280 # 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
|
|
|
2281 if ($sum_of_alignment_scores_1 == $sum_of_alignment_scores_second_best){
|
|
|
2282 $alignment_ambiguous = 1;
|
|
|
2283 # print "This read will be chucked (AS==XS detected)!\n";
|
|
|
2284
|
|
|
2285 ## need to read and discard all additional ambiguous reads until we reach the next sequence
|
|
|
2286 until ($fhs[$index]->{last_seq_id} ne $identifier){
|
|
|
2287 my $newline_1 = $fhs[$index]->{fh}-> getline();
|
|
|
2288 my $newline_2 = $fhs[$index]->{fh}-> getline();
|
|
|
2289 if ($newline_1 and $newline_2){
|
|
|
2290 chomp $newline_1;
|
|
|
2291 chomp $newline_2;
|
|
|
2292 my ($seq_id_1) = split (/\t/,$newline_1);
|
|
|
2293 my ($seq_id_2) = split (/\t/,$newline_2);
|
|
|
2294 $seq_id_1 =~ s/\/1$//;
|
|
|
2295 $seq_id_2 =~ s/\/2$//;
|
|
|
2296 # print "New Seq IDs:\t$seq_id_1\t$seq_id_2\n";
|
|
|
2297
|
|
|
2298 $fhs[$index]->{last_seq_id} = $seq_id_1;
|
|
|
2299 $fhs[$index]->{last_line_1} = $newline_1;
|
|
|
2300 $fhs[$index]->{last_line_2} = $newline_2;
|
|
|
2301 }
|
|
|
2302 else{
|
|
|
2303 # assigning undef to last_seq_id and last_line and jumping to the next index (end of Bowtie 2 output)
|
|
|
2304 $fhs[$index]->{last_seq_id} = undef;
|
|
|
2305 $fhs[$index]->{last_line_1} = undef;
|
|
|
2306 $fhs[$index]->{last_line_2} = undef;
|
|
|
2307 last; # break free if the end of the alignment output was reached
|
|
|
2308 }
|
|
|
2309 }
|
|
|
2310 # if ($fhs[$index]->{last_seq_id}){
|
|
|
2311 # warn "Index: $index\tThis Seq-ID is $identifier, skipped all ambiguous sequences until the next ID which is: $fhs[$index]->{last_seq_id}\n";
|
|
|
2312 # }
|
|
|
2313 }
|
|
|
2314 else{ # the next best alignment has a lower alignment score than the current read, so we can safely store the current alignment
|
|
|
2315
|
|
|
2316 my $alignment_location;
|
|
|
2317 if ($position_1 <= $position_2){
|
|
|
2318 $alignment_location = join(":",$chromosome_1,$position_1,$position_2);
|
|
|
2319 }
|
|
|
2320 elsif($position_2 < $position_1){
|
|
|
2321 $alignment_location = join(":",$chromosome_1,$position_2,$position_1);
|
|
|
2322 }
|
|
|
2323
|
|
|
2324 ### 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
|
|
|
2325 ### 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
|
|
|
2326 ### 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
|
|
|
2327 ### 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
|
|
|
2328
|
|
|
2329 unless (exists $alignments{$alignment_location}){
|
|
|
2330 $alignments{$alignment_location}->{seq_id} = $id_1;
|
|
|
2331 $alignments{$alignment_location}->{alignment_score_1} = $alignment_score_1;
|
|
|
2332 $alignments{$alignment_location}->{alignment_score_2} = $alignment_score_2;
|
|
|
2333 $alignments{$alignment_location}->{sum_of_alignment_scores} = $sum_of_alignment_scores_1;
|
|
|
2334 $alignments{$alignment_location}->{bowtie_sequence_1} = $bowtie_sequence_1;
|
|
|
2335 $alignments{$alignment_location}->{bowtie_sequence_2} = $bowtie_sequence_2;
|
|
|
2336 $alignments{$alignment_location}->{index} = $index;
|
|
|
2337 $alignments{$alignment_location}->{chromosome} = $chromosome_1; # either is fine
|
|
|
2338 $alignments{$alignment_location}->{position_1} = $position_1;
|
|
|
2339 $alignments{$alignment_location}->{position_2} = $position_2;
|
|
|
2340 $alignments{$alignment_location}->{mismatch_info_1} = $MD_tag_1;
|
|
|
2341 $alignments{$alignment_location}->{mismatch_info_2} = $MD_tag_2;
|
|
|
2342 $alignments{$alignment_location}->{CIGAR_1} = $cigar_1;
|
|
|
2343 $alignments{$alignment_location}->{CIGAR_2} = $cigar_2;
|
|
|
2344 $alignments{$alignment_location}->{flag_1} = $flag_1;
|
|
|
2345 $alignments{$alignment_location}->{flag_2} = $flag_2;
|
|
|
2346 }
|
|
|
2347 # warn "added best of several alignments to \%alignments hash\n";
|
|
|
2348
|
|
|
2349 ### now reading and discarding all (inferior) alignments of this read pair until we hit the next sequence
|
|
|
2350 until ($fhs[$index]->{last_seq_id} ne $identifier){
|
|
|
2351 my $newline_1 = $fhs[$index]->{fh}-> getline();
|
|
|
2352 my $newline_2 = $fhs[$index]->{fh}-> getline();
|
|
|
2353 if ($newline_1 and $newline_2){
|
|
|
2354 chomp $newline_1;
|
|
|
2355 chomp $newline_2;
|
|
|
2356 my ($seq_id_1) = split (/\t/,$newline_1);
|
|
|
2357 my ($seq_id_2) = split (/\t/,$newline_2);
|
|
|
2358 $seq_id_1 =~ s/\/1$//;
|
|
|
2359 $seq_id_2 =~ s/\/2$//;
|
|
|
2360 # print "New Seq IDs:\t$seq_id_1\t$seq_id_2\n";
|
|
|
2361
|
|
|
2362 $fhs[$index]->{last_seq_id} = $seq_id_1;
|
|
|
2363 $fhs[$index]->{last_line_1} = $newline_1;
|
|
|
2364 $fhs[$index]->{last_line_2} = $newline_2;
|
|
|
2365 }
|
|
|
2366 else{
|
|
|
2367 # assigning undef to last_seq_id and last_line_1 and _2 and jumping to the next index (end of Bowtie 2 output)
|
|
|
2368 $fhs[$index]->{last_seq_id} = undef;
|
|
|
2369 $fhs[$index]->{last_line_1} = undef;
|
|
|
2370 $fhs[$index]->{last_line_2} = undef;
|
|
|
2371 last; # break free if the end of the alignment output was reached
|
|
|
2372 }
|
|
|
2373 }
|
|
|
2374 # if($fhs[$index]->{last_seq_id}){
|
|
|
2375 # 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";
|
|
|
2376 # }
|
|
|
2377 }
|
|
|
2378 }
|
|
|
2379 else{ # there is no second best hit, so we can just store this one and read in the next sequence
|
|
|
2380
|
|
|
2381 my $alignment_location = join(":",$chromosome_1,$position_1,$position_2);
|
|
|
2382 # print "$alignment_location\n";
|
|
|
2383 ### 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
|
|
|
2384 ### 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
|
|
|
2385 ### 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
|
|
|
2386 ### 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
|
|
|
2387
|
|
|
2388 unless (exists $alignments{$alignment_location}){
|
|
|
2389 $alignments{$alignment_location}->{seq_id} = $id_1;
|
|
|
2390 $alignments{$alignment_location}->{alignment_score_1} = $alignment_score_1;
|
|
|
2391 $alignments{$alignment_location}->{alignment_score_2} = $alignment_score_2;
|
|
|
2392 $alignments{$alignment_location}->{sum_of_alignment_scores} = $sum_of_alignment_scores_1;
|
|
|
2393 $alignments{$alignment_location}->{bowtie_sequence_1} = $bowtie_sequence_1;
|
|
|
2394 $alignments{$alignment_location}->{bowtie_sequence_2} = $bowtie_sequence_2;
|
|
|
2395 $alignments{$alignment_location}->{index} = $index;
|
|
|
2396 $alignments{$alignment_location}->{chromosome} = $chromosome_1; # either is fine
|
|
|
2397 $alignments{$alignment_location}->{position_1} = $position_1;
|
|
|
2398 $alignments{$alignment_location}->{position_2} = $position_2;
|
|
|
2399 $alignments{$alignment_location}->{mismatch_info_1} = $MD_tag_1;
|
|
|
2400 $alignments{$alignment_location}->{mismatch_info_2} = $MD_tag_2;
|
|
|
2401 $alignments{$alignment_location}->{CIGAR_1} = $cigar_1;
|
|
|
2402 $alignments{$alignment_location}->{CIGAR_2} = $cigar_2;
|
|
|
2403 $alignments{$alignment_location}->{flag_1} = $flag_1;
|
|
|
2404 $alignments{$alignment_location}->{flag_2} = $flag_2;
|
|
|
2405 }
|
|
|
2406
|
|
|
2407 # warn "added unique alignment to \%alignments hash\n";
|
|
|
2408
|
|
|
2409 # Now reading and storing the next read pair
|
|
|
2410 my $newline_1 = $fhs[$index]->{fh}-> getline();
|
|
|
2411 my $newline_2 = $fhs[$index]->{fh}-> getline();
|
|
|
2412 if ($newline_1 and $newline_2){
|
|
|
2413 chomp $newline_1;
|
|
|
2414 chomp $newline_2;
|
|
|
2415 # print "$newline_1\n";
|
|
|
2416 # print "$newline_2\n";
|
|
|
2417 my ($seq_id_1) = split (/\t/,$newline_1);
|
|
|
2418 my ($seq_id_2) = split (/\t/,$newline_2);
|
|
|
2419 $seq_id_1 =~ s/\/1$//;
|
|
|
2420 $seq_id_2 =~ s/\/2$//;
|
|
|
2421 # print "New Seq IDs:\t$seq_id_1\t$seq_id_2\n";
|
|
|
2422
|
|
|
2423 $fhs[$index]->{last_seq_id} = $seq_id_1;
|
|
|
2424 $fhs[$index]->{last_line_1} = $newline_1;
|
|
|
2425 $fhs[$index]->{last_line_2} = $newline_2;
|
|
|
2426
|
|
|
2427 if ($seq_id_1 eq $identifier){
|
|
|
2428 die "Sequence with ID $identifier did not have a second best alignment, but next seq-ID was also $fhs[$index]->{last_seq_id}!\n";
|
|
|
2429 }
|
|
|
2430 }
|
|
|
2431 else{
|
|
|
2432 # assigning undef to last_seq_id and last_line_1 and _2 and jumping to the next index (end of Bowtie 2 output)
|
|
|
2433 $fhs[$index]->{last_seq_id} = undef;
|
|
|
2434 $fhs[$index]->{last_line_1} = undef;
|
|
|
2435 $fhs[$index]->{last_line_2} = undef;
|
|
|
2436 }
|
|
|
2437 }
|
|
|
2438 }
|
|
|
2439 }
|
|
|
2440
|
|
|
2441 ### 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
|
|
|
2442 if ($alignment_ambiguous == 1){
|
|
|
2443 $counting{unsuitable_sequence_count}++;
|
|
|
2444 ### 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
|
|
|
2445 # my $ambiguous_read_1 = join("\t",$identifier.'/1','256','*','0','0','*','*','0','0',$sequence_1,$quality_value_1);
|
|
|
2446 # my $ambiguous_read_2 = join("\t",$identifier.'/2','256','*','0','0','*','*','0','0',$sequence_2,$quality_value_2);
|
|
|
2447 # print "$ambiguous_read_1\n";
|
|
|
2448 # print "$ambiguous_read_2\n";
|
|
|
2449
|
|
|
2450 if ($ambiguous){
|
|
|
2451 return 2; # => exits to next sequence pair, and prints it out to _ambiguous_reads_1.txt and _ambiguous_reads_2.txt if '--ambiguous' was specified
|
|
|
2452 }
|
|
|
2453 elsif ($unmapped){
|
|
|
2454 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
|
|
|
2455 }
|
|
|
2456 else{
|
|
|
2457 return 0;
|
|
|
2458 }
|
|
|
2459 }
|
|
|
2460
|
|
|
2461 ### if no alignment was found for a certain sequence at all we continue with the next sequence in the sequence file
|
|
|
2462 unless (%alignments){
|
|
|
2463 $counting{no_single_alignment_found}++;
|
|
|
2464
|
|
|
2465 # my $unmapped_read_1 = join("\t",$identifier.'/1','77','*','0','0','*','*','0','0',$sequence_1,$quality_value_1);
|
|
|
2466 # my $unmapped_read_2 = join("\t",$identifier.'/2','141','*','0','0','*','*','0','0',$sequence_2,$quality_value_2);
|
|
|
2467 # print "$unmapped_read_1\n";
|
|
|
2468 # print "$unmapped_read_2\n";
|
|
|
2469 if ($unmapped){
|
|
|
2470 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
|
|
|
2471 }
|
|
|
2472 else{
|
|
|
2473 return 0;
|
|
|
2474 }
|
|
|
2475 }
|
|
|
2476
|
|
|
2477 #######################################################################################################################################################
|
|
|
2478
|
|
|
2479 ### 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
|
|
|
2480 ### 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)
|
|
|
2481 ### alignment score we are discarding the sequence pair altogether.
|
|
|
2482 ### 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)
|
|
|
2483 ### and extending (3 per bp) the gap.
|
|
|
2484
|
|
|
2485 #######################################################################################################################################################
|
|
|
2486
|
|
|
2487 ### Declaring an empty hash reference which will store all information we need for the methylation call
|
|
|
2488 my $methylation_call_params; # hash reference
|
|
|
2489 my $sequence_pair_fails = 0; # using $sequence_pair_fails as a 'memory' if a sequence could not be aligned uniquely (set to 1 then)
|
|
|
2490
|
|
|
2491 ### print contents of %alignments for debugging
|
|
|
2492 ## if (scalar keys %alignments >= 1){
|
|
|
2493 # print "\n******\n";
|
|
|
2494 # foreach my $alignment_location (sort {$a cmp $b} keys %alignments){
|
|
|
2495 # print "Loc: $alignment_location\n";
|
|
|
2496 # print "ID: $alignments{$alignment_location}->{seq_id}\n";
|
|
|
2497 # print "AS_1: $alignments{$alignment_location}->{alignment_score_1}\n";
|
|
|
2498 # print "AS_2: $alignments{$alignment_location}->{alignment_score_2}\n";
|
|
|
2499 # print "Seq_1: $alignments{$alignment_location}->{bowtie_sequence_1}\n";
|
|
|
2500 # print "Seq_2: $alignments{$alignment_location}->{bowtie_sequence_2}\n";
|
|
|
2501 # print "Index $alignments{$alignment_location}->{index}\n";
|
|
|
2502 # print "Chr: $alignments{$alignment_location}->{chromosome}\n";
|
|
|
2503 # print "Pos_1: $alignments{$alignment_location}->{position_1}\n";
|
|
|
2504 # print "Pos_2: $alignments{$alignment_location}->{position_2}\n";
|
|
|
2505 # print "CIGAR_1: $alignments{$alignment_location}->{CIGAR_1}\n";
|
|
|
2506 # print "CIGAR_2: $alignments{$alignment_location}->{CIGAR_2}\n";
|
|
|
2507 # print "MD_1: $alignments{$alignment_location}->{mismatch_info_1}\n";
|
|
|
2508 # print "MD_2: $alignments{$alignment_location}->{mismatch_info_2}\n";
|
|
|
2509 # print "Flag 1: $alignments{$alignment_location}->{flag_1}\n";
|
|
|
2510 # print "Flag 2: $alignments{$alignment_location}->{flag_2}\n";
|
|
|
2511 # }
|
|
|
2512 # print "\n******\n";
|
|
|
2513 # }
|
|
|
2514
|
|
|
2515 ### if there is only 1 entry in the %alignments hash we accept it as the best alignment
|
|
|
2516 if (scalar keys %alignments == 1){
|
|
|
2517 for my $unique_best_alignment (keys %alignments){
|
|
|
2518 $methylation_call_params->{$identifier}->{bowtie_sequence_1} = $alignments{$unique_best_alignment}->{bowtie_sequence_1};
|
|
|
2519 $methylation_call_params->{$identifier}->{bowtie_sequence_2} = $alignments{$unique_best_alignment}->{bowtie_sequence_2};
|
|
|
2520 $methylation_call_params->{$identifier}->{chromosome} = $alignments{$unique_best_alignment}->{chromosome};
|
|
|
2521 $methylation_call_params->{$identifier}->{position_1} = $alignments{$unique_best_alignment}->{position_1};
|
|
|
2522 $methylation_call_params->{$identifier}->{position_2} = $alignments{$unique_best_alignment}->{position_2};
|
|
|
2523 $methylation_call_params->{$identifier}->{index} = $alignments{$unique_best_alignment}->{index};
|
|
|
2524 $methylation_call_params->{$identifier}->{alignment_score_1} = $alignments{$unique_best_alignment}->{alignment_score_1};
|
|
|
2525 $methylation_call_params->{$identifier}->{alignment_score_2} = $alignments{$unique_best_alignment}->{alignment_score_2};
|
|
|
2526 $methylation_call_params->{$identifier}->{sum_of_alignment_scores} = $alignments{$unique_best_alignment}->{sum_of_alignment_scores};
|
|
|
2527 $methylation_call_params->{$identifier}->{mismatch_info_1} = $alignments{$unique_best_alignment}->{mismatch_info_1};
|
|
|
2528 $methylation_call_params->{$identifier}->{mismatch_info_2} = $alignments{$unique_best_alignment}->{mismatch_info_2};
|
|
|
2529 $methylation_call_params->{$identifier}->{CIGAR_1} = $alignments{$unique_best_alignment}->{CIGAR_1};
|
|
|
2530 $methylation_call_params->{$identifier}->{CIGAR_2} = $alignments{$unique_best_alignment}->{CIGAR_2};
|
|
|
2531 $methylation_call_params->{$identifier}->{flag_1} = $alignments{$unique_best_alignment}->{flag_1};
|
|
|
2532 $methylation_call_params->{$identifier}->{flag_2} = $alignments{$unique_best_alignment}->{flag_2};
|
|
|
2533 }
|
|
|
2534 }
|
|
|
2535
|
|
|
2536 ### 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
|
|
|
2537 ### we boot the sequence pair altogether)
|
|
|
2538 elsif (scalar keys %alignments >= 2 and scalar keys %alignments <= 4){
|
|
|
2539 my $best_sum_of_alignment_scores;
|
|
|
2540 my $best_alignment_location;
|
|
|
2541 foreach my $alignment_location (sort {$alignments{$b}->{sum_of_alignment_scores} <=> $alignments{$a}->{sum_of_alignment_scores}} keys %alignments){
|
|
|
2542 # print "$alignments{$alignment_location}->{sum_of_alignment_scores}\n";
|
|
|
2543 unless (defined $best_sum_of_alignment_scores){
|
|
|
2544 $best_sum_of_alignment_scores = $alignments{$alignment_location}->{sum_of_alignment_scores};
|
|
|
2545 $best_alignment_location = $alignment_location;
|
|
|
2546 # print "setting best alignment score to: $best_sum_of_alignment_scores\n";
|
|
|
2547 }
|
|
|
2548 else{
|
|
|
2549 ### if the second best alignment has the same sum of alignment scores as the first one, the sequence pair will get booted
|
|
|
2550 if ($alignments{$alignment_location}->{sum_of_alignment_scores} == $best_sum_of_alignment_scores){
|
|
|
2551 # warn "Same sum of alignment scores for 2 different alignments, the sequence pair will get booted!\n";
|
|
|
2552 $sequence_pair_fails = 1;
|
|
|
2553 last; # exiting since we know that the sequence has ambiguous alignments
|
|
|
2554 }
|
|
|
2555 ### else we are going to store the best alignment for further processing
|
|
|
2556 else{
|
|
|
2557 $methylation_call_params->{$identifier}->{bowtie_sequence_1} = $alignments{$best_alignment_location}->{bowtie_sequence_1};
|
|
|
2558 $methylation_call_params->{$identifier}->{bowtie_sequence_2} = $alignments{$best_alignment_location}->{bowtie_sequence_2};
|
|
|
2559 $methylation_call_params->{$identifier}->{chromosome} = $alignments{$best_alignment_location}->{chromosome};
|
|
|
2560 $methylation_call_params->{$identifier}->{position_1} = $alignments{$best_alignment_location}->{position_1};
|
|
|
2561 $methylation_call_params->{$identifier}->{position_2} = $alignments{$best_alignment_location}->{position_2};
|
|
|
2562 $methylation_call_params->{$identifier}->{index} = $alignments{$best_alignment_location}->{index};
|
|
|
2563 $methylation_call_params->{$identifier}->{alignment_score_1} = $alignments{$best_alignment_location}->{alignment_score_1};
|
|
|
2564 $methylation_call_params->{$identifier}->{alignment_score_2} = $alignments{$best_alignment_location}->{alignment_score_2};
|
|
|
2565 $methylation_call_params->{$identifier}->{sum_of_alignment_scores} = $alignments{$best_alignment_location}->{sum_of_alignment_scores};
|
|
|
2566 $methylation_call_params->{$identifier}->{mismatch_info_1} = $alignments{$best_alignment_location}->{mismatch_info_1};
|
|
|
2567 $methylation_call_params->{$identifier}->{mismatch_info_2} = $alignments{$best_alignment_location}->{mismatch_info_2};
|
|
|
2568 $methylation_call_params->{$identifier}->{CIGAR_1} = $alignments{$best_alignment_location}->{CIGAR_1};
|
|
|
2569 $methylation_call_params->{$identifier}->{CIGAR_2} = $alignments{$best_alignment_location}->{CIGAR_2};
|
|
|
2570 $methylation_call_params->{$identifier}->{flag_1} = $alignments{$best_alignment_location}->{flag_1};
|
|
|
2571 $methylation_call_params->{$identifier}->{flag_2} = $alignments{$best_alignment_location}->{flag_2};
|
|
|
2572 last; # exiting since the sequence produced a unique best alignment
|
|
|
2573 }
|
|
|
2574 }
|
|
|
2575 }
|
|
|
2576 }
|
|
|
2577 else{
|
|
|
2578 die "There are too many potential hits for this sequence pair (1-4 expected, but found: '",scalar keys %alignments,"')\n";;
|
|
|
2579 }
|
|
|
2580
|
|
|
2581 ### skipping the sequence completely if there were multiple alignments with the same best sum of alignment scores at different positions
|
|
|
2582 if ($sequence_pair_fails == 1){
|
|
|
2583 $counting{unsuitable_sequence_count}++;
|
|
|
2584
|
|
|
2585 ### 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
|
|
|
2586 # my $ambiguous_read_1 = join("\t",$identifier.'/1','256','*','0','0','*','*','0','0',$sequence_1,$quality_value_1);
|
|
|
2587 # my $ambiguous_read_2 = join("\t",$identifier.'/2','256','*','0','0','*','*','0','0',$sequence_2,$quality_value_2);
|
|
|
2588 # print "$ambiguous_read_1\n";
|
|
|
2589 # print "$ambiguous_read_2\n";
|
|
|
2590
|
|
|
2591 if ($ambiguous){
|
|
|
2592 return 2; # => exits to next sequence pair, and prints it out (in FastQ format) to _ambiguous_reads_1.txt and _ambiguous_reads_2.txt if '--ambiguous' was specified
|
|
|
2593 }
|
|
|
2594 elsif ($unmapped){
|
|
|
2595 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
|
|
|
2596 }
|
|
|
2597 else{
|
|
|
2598 return 0; # => exits to next sequence pair (default)
|
|
|
2599 }
|
|
|
2600 }
|
|
|
2601
|
|
|
2602 ### --DIRECTIONAL
|
|
|
2603 ### 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
|
|
|
2604 ### discard all alignments to strands complementary to the original strands, as they should not exist in reality due to the library preparation protocol
|
|
|
2605 if ($directional){
|
|
|
2606 if ( ($methylation_call_params->{$identifier}->{index} == 1) or ($methylation_call_params->{$identifier}->{index} == 2) ){
|
|
|
2607 # warn "Alignment rejected! (index was: $methylation_call_params->{$identifier}->{index})\n";
|
|
|
2608 $counting{alignments_rejected_count}++;
|
|
|
2609 return 0;
|
|
|
2610 }
|
|
|
2611 }
|
|
|
2612
|
|
|
2613 ### If the sequence pair has not been rejected so far it does have a unique best alignment
|
|
|
2614 $counting{unique_best_alignment_count}++;
|
|
|
2615 extract_corresponding_genomic_sequence_paired_ends_bowtie2($identifier,$methylation_call_params);
|
|
|
2616
|
|
|
2617 ### check to see if the genomic sequences we extracted has the same length as the observed sequences +2, and only then we perform the methylation call
|
|
|
2618 if (length($methylation_call_params->{$identifier}->{unmodified_genomic_sequence_1}) != length($sequence_1)+2){
|
|
|
2619 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";
|
|
|
2620 $counting{genomic_sequence_could_not_be_extracted_count}++;
|
|
|
2621 return 0;
|
|
|
2622 }
|
|
|
2623 if (length($methylation_call_params->{$identifier}->{unmodified_genomic_sequence_2}) != length($sequence_2)+2){
|
|
|
2624 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";
|
|
|
2625 $counting{genomic_sequence_could_not_be_extracted_count}++;
|
|
|
2626 return 0;
|
|
|
2627 }
|
|
|
2628
|
|
|
2629 ### now we are set to perform the actual methylation call
|
|
|
2630 $methylation_call_params->{$identifier}->{methylation_call_1} = methylation_call($identifier,$sequence_1,$methylation_call_params->{$identifier}->{unmodified_genomic_sequence_1},$methylation_call_params->{$identifier}->{read_conversion_1});
|
|
|
2631 $methylation_call_params->{$identifier}->{methylation_call_2} = methylation_call($identifier,$sequence_2,$methylation_call_params->{$identifier}->{unmodified_genomic_sequence_2},$methylation_call_params->{$identifier}->{read_conversion_2});
|
|
|
2632 # print "$methylation_call_params->{$identifier}->{read_conversion_2}\n";
|
|
|
2633 # print " $sequence_2\n";
|
|
|
2634 # print "$methylation_call_params->{$identifier}->{unmodified_genomic_sequence_2}\n";
|
|
|
2635 # print " $methylation_call_params->{$identifier}->{methylation_call_2}\n";
|
|
|
2636
|
|
|
2637 print_bisulfite_mapping_results_paired_ends_bowtie2($identifier,$sequence_1,$sequence_2,$methylation_call_params,$quality_value_1,$quality_value_2);
|
|
|
2638 return 0; ## otherwise 1 will be returned by default, which would print the sequence pair to unmapped_1 and _2
|
|
|
2639 }
|
|
|
2640
|
|
|
2641 ###
|
|
|
2642
|
|
|
2643 sub decide_whether_paired_end_alignment_is_valid{
|
|
|
2644 my ($index,$identifier) = @_;
|
|
|
2645 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];
|
|
|
2646 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];
|
|
|
2647 chomp $mismatch_info_1;
|
|
|
2648 chomp $mismatch_info_2;
|
|
|
2649 my $seq_id_1 = $id_1;
|
|
|
2650 my $seq_id_2 = $id_2;
|
|
|
2651 $seq_id_1 =~ s/\/1$//; # removing the read /1
|
|
|
2652 $seq_id_2 =~ s/\/1$//; # removing the read /1
|
|
|
2653
|
|
|
2654 ### ensuring that the current entry is the correct sequence
|
|
|
2655 if ($seq_id_1 eq $identifier or $seq_id_2 eq $identifier){
|
|
|
2656 ### checking the orientation of the alignment. We need to discriminate between 8 different conditions, however only 4 of them are theoretically
|
|
|
2657 ### sensible alignments
|
|
|
2658 my $orientation = ensure_sensical_alignment_orientation_paired_ends ($index,$id_1,$strand_1,$id_2,$strand_2);
|
|
|
2659 ### If the orientation was correct can we move on
|
|
|
2660 if ($orientation == 1){
|
|
|
2661 return 1; ### 1st possibility for A SEQUENCE-PAIR TO PASS
|
|
|
2662 }
|
|
|
2663 ### If the alignment was in the wrong orientation we need to read in two new lines
|
|
|
2664 elsif($orientation == 0){
|
|
|
2665 my $newline_1 = $fhs[$index]->{fh}->getline();
|
|
|
2666 my $newline_2 = $fhs[$index]->{fh}->getline();
|
|
|
2667 if ($newline_1 and $newline_2){
|
|
|
2668 ### extract detailed information about the alignment again (from $newline_1 and $newline_2 this time)
|
|
|
2669 ($id_1,$strand_1) = (split (/\t/,$newline_1))[0,1];
|
|
|
2670 ($id_2,$strand_2) = (split (/\t/,$newline_2))[0,1];
|
|
|
2671
|
|
|
2672 my $seqid;
|
|
|
2673 $seq_id_1 = $id_1;
|
|
|
2674 $seq_id_2 = $id_2;
|
|
|
2675 # we need to capture the first read (ending on /1)
|
|
|
2676 if ($seq_id_1 =~ s/\/1$//){ # removing the read /1 tag
|
|
|
2677 $seqid = $seq_id_1;
|
|
|
2678 }
|
|
|
2679 elsif ($seq_id_2 =~ s/\/1$//){ # removing the read /1 tag
|
|
|
2680 $seqid = $seq_id_2;
|
|
|
2681 }
|
|
|
2682 else{
|
|
|
2683 die "One of the two reads needs to end on /1!!";
|
|
|
2684 }
|
|
|
2685
|
|
|
2686 ### ensuring that the next entry is still the correct sequence
|
|
|
2687 if ($seq_id_1 eq $identifier or $seq_id_2 eq $identifier){
|
|
|
2688 ### checking orientation again
|
|
|
2689 $orientation = ensure_sensical_alignment_orientation_paired_ends ($index,$id_1,$strand_1,$id_2,$strand_2);
|
|
|
2690 ### If the orientation was correct can we move on
|
|
|
2691 if ($orientation == 1){
|
|
|
2692 ### Writing the current sequence to last_line_1 and last_line_2
|
|
|
2693 $fhs[$index]->{last_seq_id} = $seqid;
|
|
|
2694 $fhs[$index]->{last_line_1} = $newline_1;
|
|
|
2695 $fhs[$index]->{last_line_2} = $newline_2;
|
|
|
2696 return 1; ### 2nd possibility for a SEQUENCE-PAIR TO PASS
|
|
|
2697 }
|
|
|
2698 ### If the alignment was in the wrong orientation again we need to read in yet another 2 new lines and store them in @fhs (this must be
|
|
|
2699 ### the next entry)
|
|
|
2700 elsif ($orientation == 0){
|
|
|
2701 $newline_1 = $fhs[$index]->{fh}->getline();
|
|
|
2702 $newline_2 = $fhs[$index]->{fh}->getline();
|
|
|
2703 if ($newline_1 and $newline_2){
|
|
|
2704 ($seq_id_1) = split (/\t/,$newline_1);
|
|
|
2705 ($seq_id_2) = split (/\t/,$newline_2);
|
|
|
2706
|
|
|
2707 $seqid = '';
|
|
|
2708 if ($seq_id_1 =~ s/\/1$//){ # removing the read /1 tag
|
|
|
2709 $seqid = $seq_id_1;
|
|
|
2710 }
|
|
|
2711 elsif ($seq_id_2 =~ s/\/1$//){ # removing the read /1 tag
|
|
|
2712 $seqid = $seq_id_2;
|
|
|
2713 }
|
|
|
2714 else{
|
|
|
2715 die "One of the two reads needs to end on /1!!";
|
|
|
2716 }
|
|
|
2717
|
|
|
2718 ### check if the next 2 lines still have the same seq ID (must not happen), and if not overwrite the current seq-ID and bowtie output with
|
|
|
2719 ### the same fields of the just read next entry
|
|
|
2720 die "Same seq ID 3 or more times in a row!(should be 2 max)" if ($seqid eq $identifier);
|
|
|
2721 $fhs[$index]->{last_seq_id} = $seqid;
|
|
|
2722 $fhs[$index]->{last_line_1} = $newline_1;
|
|
|
2723 $fhs[$index]->{last_line_2} = $newline_2;
|
|
|
2724 return 0; # not processing anything this round as the alignment currently stored in last_line_1 and _2 was in the wrong orientation
|
|
|
2725 }
|
|
|
2726 else {
|
|
|
2727 ### assigning undef to last_seq_id and last_line (end of bowtie output)
|
|
|
2728 $fhs[$index]->{last_seq_id} = undef;
|
|
|
2729 $fhs[$index]->{last_line_1} = undef;
|
|
|
2730 $fhs[$index]->{last_line_2} = undef;
|
|
|
2731 return 0; # not processing anything as the alignment currently stored in last_line_1 and _2 was in the wrong orientation
|
|
|
2732 }
|
|
|
2733 }
|
|
|
2734 else{
|
|
|
2735 die "The orientation of the alignment must be either correct or incorrect\n";
|
|
|
2736 }
|
|
|
2737 }
|
|
|
2738 ### the sequence pair we just read in is already the next sequence pair to be analysed -> store it in @fhs
|
|
|
2739 else{
|
|
|
2740 $fhs[$index]->{last_seq_id} = $seqid;
|
|
|
2741 $fhs[$index]->{last_line_1} = $newline_1;
|
|
|
2742 $fhs[$index]->{last_line_2} = $newline_2;
|
|
|
2743 return 0; # processing the new alignment result only in the next round
|
|
|
2744 }
|
|
|
2745 }
|
|
|
2746 else {
|
|
|
2747 # assigning undef to last_seq_id and both last_lines (end of bowtie output)
|
|
|
2748 $fhs[$index]->{last_seq_id} = undef;
|
|
|
2749 $fhs[$index]->{last_line_1} = undef;
|
|
|
2750 $fhs[$index]->{last_line_2} = undef;
|
|
|
2751 return 0; # not processing anything as the alignment currently stored in last_line_1 and _2 was in the wrong orientation
|
|
|
2752 }
|
|
|
2753 }
|
|
|
2754 else{
|
|
|
2755 die "The orientation of the alignment must be either correct or incorrect\n";
|
|
|
2756 }
|
|
|
2757 }
|
|
|
2758 ### 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
|
|
|
2759 else{
|
|
|
2760 return 0;
|
|
|
2761 }
|
|
|
2762 }
|
|
|
2763
|
|
|
2764 ### EXTRACT GENOMIC SEQUENCE | BOWTIE 1 | PAIRED-END
|
|
|
2765
|
|
|
2766 sub extract_corresponding_genomic_sequence_paired_ends {
|
|
|
2767 my ($sequence_identifier,$methylation_call_params) = @_;
|
|
|
2768 ### 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
|
|
|
2769 ### sequence a 'memory' of the conversion we are expecting which we will need later for the methylation call
|
|
|
2770 my $alignment_read_1;
|
|
|
2771 my $alignment_read_2;
|
|
|
2772 my $read_conversion_info_1;
|
|
|
2773 my $read_conversion_info_2;
|
|
|
2774 my $genome_conversion;
|
|
|
2775
|
|
|
2776 ### 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
|
|
|
2777 ### if the C happens to be at the first or last position of the actually observed sequence
|
|
|
2778 my $non_bisulfite_sequence_1;
|
|
|
2779 my $non_bisulfite_sequence_2;
|
|
|
2780
|
|
|
2781 ### all alignments reported by bowtie have the + alignment first and the - alignment as the second one irrespective of whether read 1 or read 2 was
|
|
|
2782 ### the + alignment. We however always read in sequences read 1 then read 2, so if read 2 is the + alignment we need to swap the extracted genomic
|
|
|
2783 ### sequences around!
|
|
|
2784 ### results from CT converted read 1 plus GA converted read 2 vs. CT converted genome (+/- orientation alignments are reported only)
|
|
|
2785 if ($methylation_call_params->{$sequence_identifier}->{index} == 0){
|
|
|
2786 ### [Index 0, sequence originated from (converted) forward strand]
|
|
|
2787 $counting{CT_GA_CT_count}++;
|
|
|
2788 $alignment_read_1 = '+';
|
|
|
2789 $alignment_read_2 = '-';
|
|
|
2790 $read_conversion_info_1 = 'CT';
|
|
|
2791 $read_conversion_info_2 = 'GA';
|
|
|
2792 $genome_conversion = 'CT';
|
|
|
2793 ### SEQUENCE 1 (this is always the forward hit, in this case it is read 1)
|
|
|
2794 ### for hits on the forward strand we need to capture 2 extra bases at the 3' end
|
|
|
2795
|
|
|
2796 $non_bisulfite_sequence_1 = substr ($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
|
|
|
2797
|
|
|
2798 ### SEQUENCE 2 (this will always be on the reverse strand, in this case it is read 2)
|
|
|
2799 ### As the second conversion is GA we need to capture 1 base 3', so that it is a 5' base after reverse complementation
|
|
|
2800 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
|
|
|
2801
|
|
|
2802 $non_bisulfite_sequence_2 = substr ($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);
|
|
|
2803 ### the reverse strand sequence needs to be reverse complemented
|
|
|
2804 $non_bisulfite_sequence_2 = reverse_complement($non_bisulfite_sequence_2);
|
|
|
2805 }
|
|
|
2806 else{
|
|
|
2807 $non_bisulfite_sequence_2 = '';
|
|
|
2808 }
|
|
|
2809 }
|
|
|
2810
|
|
|
2811 ### results from GA converted read 1 plus CT converted read 2 vs. GA converted genome (+/- orientation alignments are reported only)
|
|
|
2812 elsif ($methylation_call_params->{$sequence_identifier}->{index} == 1){
|
|
|
2813 ### [Index 1, sequence originated from complementary to (converted) reverse strand]
|
|
|
2814 $counting{GA_CT_GA_count}++;
|
|
|
2815 $alignment_read_1 = '+';
|
|
|
2816 $alignment_read_2 = '-';
|
|
|
2817 $read_conversion_info_1 = 'GA';
|
|
|
2818 $read_conversion_info_2 = 'CT';
|
|
|
2819 $genome_conversion = 'GA';
|
|
|
2820
|
|
|
2821 ### SEQUENCE 1 (this is always the forward hit, in this case it is read 1)
|
|
|
2822 ### as we need to make the methylation call for the base 5' of the first base (GA conversion!) we need to capture 2 extra bases at the 5' end
|
|
|
2823 if ($methylation_call_params->{$sequence_identifier}->{start_seq_1}-1 > 0){ ## CHH change to -1
|
|
|
2824 $non_bisulfite_sequence_1 = substr ($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
|
|
|
2825 }
|
|
|
2826 else{
|
|
|
2827 $non_bisulfite_sequence_1 = '';
|
|
|
2828 }
|
|
|
2829
|
|
|
2830 ### SEQUENCE 2 (this will always be on the reverse strand, in this case it is read 2)
|
|
|
2831 ### 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
|
|
|
2832 $non_bisulfite_sequence_2 = substr ($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
|
|
|
2833 ### the reverse strand sequence needs to be reverse complemented
|
|
|
2834 $non_bisulfite_sequence_2 = reverse_complement($non_bisulfite_sequence_2);
|
|
|
2835 }
|
|
|
2836
|
|
|
2837 ### results from GA converted read 1 plus CT converted read 2 vs. CT converted genome (-/+ orientation alignments are reported only)
|
|
|
2838 elsif ($methylation_call_params->{$sequence_identifier}->{index} == 2){
|
|
|
2839 ### [Index 2, sequence originated from the complementary to (converted) forward strand]
|
|
|
2840 $counting{GA_CT_CT_count}++;
|
|
|
2841 $alignment_read_1 = '-';
|
|
|
2842 $alignment_read_2 = '+';
|
|
|
2843 $read_conversion_info_1 = 'GA';
|
|
|
2844 $read_conversion_info_2 = 'CT';
|
|
|
2845 $genome_conversion = 'CT';
|
|
|
2846
|
|
|
2847 ### Here we switch the sequence information round!! non_bisulfite_sequence_1 will later correspond to the read 1!!!!
|
|
|
2848 ### SEQUENCE 1 (this is always the forward hit, in this case it is READ 2), read 1 is in - orientation on the reverse strand
|
|
|
2849 ### As read 1 is GA converted we need to capture 2 extra 3' bases which will be 2 extra 5' base after reverse complementation
|
|
|
2850 $non_bisulfite_sequence_1 = substr ($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
|
|
|
2851 ### the reverse strand sequence needs to be reverse complemented
|
|
|
2852 $non_bisulfite_sequence_1 = reverse_complement($non_bisulfite_sequence_1);
|
|
|
2853
|
|
|
2854 ### SEQUENCE 2 (this will always be on the reverse strand, in this case it is READ 1)
|
|
|
2855 ### non_bisulfite_sequence_2 will later correspond to the read 2!!!!
|
|
|
2856 ### Read 2 is CT converted so we need to capture 2 extra 3' bases
|
|
|
2857 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
|
|
|
2858 $non_bisulfite_sequence_2 = substr ($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
|
|
|
2859 }
|
|
|
2860 else{
|
|
|
2861 $non_bisulfite_sequence_2 = '';
|
|
|
2862 }
|
|
|
2863 }
|
|
|
2864
|
|
|
2865 ### results from CT converted read 1 plus GA converted read 2 vs. GA converted genome (-/+ orientation alignments are reported only)
|
|
|
2866 elsif ($methylation_call_params->{$sequence_identifier}->{index} == 3){
|
|
|
2867 ### [Index 3, sequence originated from the (converted) reverse strand]
|
|
|
2868 $counting{CT_GA_GA_count}++;
|
|
|
2869 $alignment_read_1 = '-';
|
|
|
2870 $alignment_read_2 = '+';
|
|
|
2871 $read_conversion_info_1 = 'CT';
|
|
|
2872 $read_conversion_info_2 = 'GA';
|
|
|
2873 $genome_conversion = 'GA';
|
|
|
2874
|
|
|
2875 ### Here we switch the sequence information round!! non_bisulfite_sequence_1 will later correspond to the read 1!!!!
|
|
|
2876 ### SEQUENCE 1 (this is always the forward hit, in this case it is READ 2), read 1 is in - orientation on the reverse strand
|
|
|
2877 ### As read 1 is CT converted we need to capture 2 extra 5' bases which will be 2 extra 3' base after reverse complementation
|
|
|
2878 if ( ($methylation_call_params->{$sequence_identifier}->{start_seq_2}-1) > 0){ ## CHH changed to -1
|
|
|
2879 $non_bisulfite_sequence_1 = substr ($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
|
|
|
2880 ### the reverse strand sequence needs to be reverse complemented
|
|
|
2881 $non_bisulfite_sequence_1 = reverse_complement($non_bisulfite_sequence_1);
|
|
|
2882 }
|
|
|
2883 else{
|
|
|
2884 $non_bisulfite_sequence_1 = '';
|
|
|
2885 }
|
|
|
2886
|
|
|
2887 ### SEQUENCE 2 (this will always be on the reverse strand, in this case it is READ 1)
|
|
|
2888 ### non_bisulfite_sequence_2 will later correspond to the read 2!!!!
|
|
|
2889 ### Read 2 is GA converted so we need to capture 2 extra 5' bases
|
|
|
2890 $non_bisulfite_sequence_2 = substr ($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
|
|
|
2891 }
|
|
|
2892 else{
|
|
|
2893 die "Too many bowtie result filehandles\n";
|
|
|
2894 }
|
|
|
2895 ### the alignment_strand information is needed to determine which strand of the genomic sequence we are comparing the read against,
|
|
|
2896 ### the read_conversion information is needed to know whether we are looking for C->T or G->A substitutions
|
|
|
2897
|
|
|
2898 $methylation_call_params->{$sequence_identifier}->{alignment_read_1} = $alignment_read_1;
|
|
|
2899 $methylation_call_params->{$sequence_identifier}->{alignment_read_2} = $alignment_read_2;
|
|
|
2900 $methylation_call_params->{$sequence_identifier}->{genome_conversion} = $genome_conversion;
|
|
|
2901 $methylation_call_params->{$sequence_identifier}->{read_conversion_1} = $read_conversion_info_1;
|
|
|
2902 $methylation_call_params->{$sequence_identifier}->{read_conversion_2} = $read_conversion_info_2;
|
|
|
2903 $methylation_call_params->{$sequence_identifier}->{unmodified_genomic_sequence_1} = $non_bisulfite_sequence_1;
|
|
|
2904 $methylation_call_params->{$sequence_identifier}->{unmodified_genomic_sequence_2} = $non_bisulfite_sequence_2;
|
|
|
2905 }
|
|
|
2906
|
|
|
2907 ### EXTRACT GENOMIC SEQUENCE BOWTIE 2 | PAIRED-END
|
|
|
2908
|
|
|
2909 sub extract_corresponding_genomic_sequence_paired_ends_bowtie2{
|
|
|
2910 my ($sequence_identifier,$methylation_call_params) = @_;
|
|
|
2911 ### 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
|
|
|
2912 ### sequence a 'memory' of the conversion we are expecting which we will need later for the methylation call
|
|
|
2913
|
|
|
2914 my $cigar_1 = $methylation_call_params->{$sequence_identifier}->{CIGAR_1};
|
|
|
2915 my $cigar_2 = $methylation_call_params->{$sequence_identifier}->{CIGAR_2};
|
|
|
2916 my $flag_1 = $methylation_call_params->{$sequence_identifier}->{flag_1};
|
|
|
2917 my $flag_2 = $methylation_call_params->{$sequence_identifier}->{flag_2};
|
|
|
2918 # print "$cigar_1\t$cigar_2\t$flag_1\t$flag_2\n";
|
|
|
2919 ### 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
|
|
|
2920 ### 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
|
|
|
2921
|
|
|
2922 ### the alignment_strand information is needed to determine which strand of the genomic sequence we are comparing the read against,
|
|
|
2923 ### the read_conversion information is needed to know whether we are looking for C->T or G->A substitutions
|
|
|
2924 my $alignment_read_1;
|
|
|
2925 my $alignment_read_2;
|
|
|
2926 my $read_conversion_info_1;
|
|
|
2927 my $read_conversion_info_2;
|
|
|
2928 my $genome_conversion;
|
|
|
2929
|
|
|
2930 ### 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
|
|
|
2931 ### if the C happens to be at the last position of the actually observed sequence
|
|
|
2932 my $non_bisulfite_sequence_1 = '';
|
|
|
2933 my $non_bisulfite_sequence_2 = '';
|
|
|
2934
|
|
|
2935 ### Positions in SAM format are 1 based, so we need to subract 1 when getting substrings
|
|
|
2936 my $pos_1 = $methylation_call_params->{$sequence_identifier}->{position_1}-1;
|
|
|
2937 my $pos_2 = $methylation_call_params->{$sequence_identifier}->{position_2}-1;
|
|
|
2938
|
|
|
2939 # parsing CIGAR 1 string
|
|
|
2940 my @len_1 = split (/\D+/,$cigar_1); # storing the length per operation
|
|
|
2941 my @ops_1 = split (/\d+/,$cigar_1); # storing the operation
|
|
|
2942 shift @ops_1; # remove the empty first element
|
|
|
2943 die "CIGAR 1 string contained a non-matching number of lengths and operations\n" unless (scalar @len_1 == scalar @ops_1);
|
|
|
2944 # parsing CIGAR 2 string
|
|
|
2945 my @len_2 = split (/\D+/,$cigar_2); # storing the length per operation
|
|
|
2946 my @ops_2 = split (/\d+/,$cigar_2); # storing the operation
|
|
|
2947 shift @ops_2; # remove the empty first element
|
|
|
2948 die "CIGAR 2 string contained a non-matching number of lengths and operations\n" unless (scalar @len_2 == scalar @ops_2);
|
|
|
2949
|
|
|
2950 my $indels_1 = 0; # addiong these to the hemming distance value (needed for the NM field in the final SAM output
|
|
|
2951 my $indels_2 = 0;
|
|
|
2952
|
|
|
2953 ### Extracting read 1 genomic sequence ###
|
|
|
2954
|
|
|
2955 # extracting 2 additional bp at the 5' end (read 1)
|
|
|
2956 if ( ($methylation_call_params->{$sequence_identifier}->{index} == 1) or ($methylation_call_params->{$sequence_identifier}->{index} == 3) ){
|
|
|
2957 # checking if the substring will be valid or if we can't extract the sequence because we are right at the edge of a chromosome
|
|
|
2958 unless ( ($pos_1-2) > 0){# exiting with en empty genomic sequence otherwise
|
|
|
2959 $methylation_call_params->{$sequence_identifier}->{unmodified_genomic_sequence_1} = $non_bisulfite_sequence_1;
|
|
|
2960 return;
|
|
|
2961 }
|
|
|
2962 $non_bisulfite_sequence_1 .= substr ($chromosomes{$methylation_call_params->{$sequence_identifier}->{chromosome}},$pos_1-2,2);
|
|
|
2963 }
|
|
|
2964
|
|
|
2965 foreach (0..$#len_1){
|
|
|
2966 if ($ops_1[$_] eq 'M'){
|
|
|
2967 # extracting genomic sequence
|
|
|
2968 $non_bisulfite_sequence_1 .= substr ($chromosomes{$methylation_call_params->{$sequence_identifier}->{chromosome}},$pos_1,$len_1[$_]);
|
|
|
2969 # warn "$non_bisulfite_sequence_1\n";
|
|
|
2970 # adjusting position
|
|
|
2971 $pos_1 += $len_1[$_];
|
|
|
2972 }
|
|
|
2973 elsif ($ops_1[$_] eq 'I'){ # insertion in the read sequence
|
|
|
2974 # we simply add padding Ns instead of finding genomic sequence. This will not be used to infer methylation calls
|
|
|
2975 $non_bisulfite_sequence_1 .= 'N' x $len_1[$_];
|
|
|
2976 # warn "$non_bisulfite_sequence_1\n";
|
|
|
2977 # position doesn't need adjusting
|
|
|
2978 $indels_1 += $len_1[$_]; # adding to $indels_1 to determine the hemming distance (= single base mismatches, insertions or deletions) for the SAM output
|
|
|
2979 }
|
|
|
2980 elsif ($ops_1[$_] eq 'D'){ # deletion in the read sequence
|
|
|
2981 # we do not add any genomic sequence but only adjust the position
|
|
|
2982 # warn "Just adjusting the position by: ",$len_1[$_],"bp\n";
|
|
|
2983 $pos_1 += $len_1[$_];
|
|
|
2984 $indels_1 += $len_1[$_]; # adding to $indels_1 to determine the hemming distance (= single base mismatches, insertions or deletions) for the SAM output
|
|
|
2985 }
|
|
|
2986 elsif($cigar_1 =~ tr/[NSHPX=]//){ # if these (for standard mapping) illegal characters exist we die
|
|
|
2987 die "The CIGAR 1 string contained illegal CIGAR operations in addition to 'M', 'I' and 'D': $cigar_1\n";
|
|
|
2988 }
|
|
|
2989 else{
|
|
|
2990 die "The CIGAR 1 string contained undefined CIGAR operations in addition to 'M', 'I' and 'D': $cigar_1\n";
|
|
|
2991 }
|
|
|
2992 }
|
|
|
2993
|
|
|
2994 ### 3' end of read 1
|
|
|
2995 if ( ($methylation_call_params->{$sequence_identifier}->{index} == 0) or ($methylation_call_params->{$sequence_identifier}->{index} == 2) ){
|
|
|
2996 ## checking if the substring will be valid or if we can't extract the sequence because we are right at the edge of a chromosome
|
|
|
2997 unless (length($chromosomes{$methylation_call_params->{$sequence_identifier}->{chromosome}}) >= $pos_1+2){# exiting with en empty genomic sequence otherwise
|
|
|
2998 $methylation_call_params->{$sequence_identifier}->{unmodified_genomic_sequence_1} = $non_bisulfite_sequence_1;
|
|
|
2999 return;
|
|
|
3000 }
|
|
|
3001 $non_bisulfite_sequence_1 .= substr ($chromosomes{$methylation_call_params->{$sequence_identifier}->{chromosome}},$pos_1,2);
|
|
|
3002 }
|
|
|
3003
|
|
|
3004
|
|
|
3005 ### Extracting read 2 genomic sequence ###
|
|
|
3006
|
|
|
3007 ### 5' end of read 2
|
|
|
3008 if ( ($methylation_call_params->{$sequence_identifier}->{index} == 1) or ($methylation_call_params->{$sequence_identifier}->{index} == 3) ){
|
|
|
3009 ## checking if the substring will be valid or if we can't extract the sequence because we are right at the edge of a chromosome
|
|
|
3010 unless ( ($pos_2-2) >= 0){# exiting with en empty genomic sequence otherwise
|
|
|
3011 $methylation_call_params->{$sequence_identifier}->{unmodified_genomic_sequence_2} = $non_bisulfite_sequence_2;
|
|
|
3012 return;
|
|
|
3013 }
|
|
|
3014 $non_bisulfite_sequence_2 .= substr ($chromosomes{$methylation_call_params->{$sequence_identifier}->{chromosome}},$pos_2-2,2);
|
|
|
3015 }
|
|
|
3016
|
|
|
3017 foreach (0..$#len_2){
|
|
|
3018 if ($ops_2[$_] eq 'M'){
|
|
|
3019 # extracting genomic sequence
|
|
|
3020 $non_bisulfite_sequence_2 .= substr ($chromosomes{$methylation_call_params->{$sequence_identifier}->{chromosome}},$pos_2,$len_2[$_]);
|
|
|
3021 # warn "$non_bisulfite_sequence_2\n";
|
|
|
3022 # adjusting position
|
|
|
3023 $pos_2 += $len_2[$_];
|
|
|
3024 }
|
|
|
3025 elsif ($ops_2[$_] eq 'I'){ # insertion in the read sequence
|
|
|
3026 # we simply add padding Ns instead of finding genomic sequence. This will not be used to infer methylation calls
|
|
|
3027 $non_bisulfite_sequence_2 .= 'N' x $len_2[$_];
|
|
|
3028 # warn "$non_bisulfite_sequence_2\n";
|
|
|
3029 # position doesn't need adjusting
|
|
|
3030 $indels_2 += $len_2[$_]; # adding to $indels_1 to determine the hemming distance (= single base mismatches, insertions or deletions) for the SAM output
|
|
|
3031 }
|
|
|
3032 elsif ($ops_2[$_] eq 'D'){ # deletion in the read sequence
|
|
|
3033 # we do not add any genomic sequence but only adjust the position
|
|
|
3034 # warn "Just adjusting the position by: ",$len_2[$_],"bp\n";
|
|
|
3035 $pos_2 += $len_2[$_];
|
|
|
3036 $indels_2 += $len_2[$_]; # adding to $indels_1 to determine the hemming distance (= single base mismatches, insertions or deletions) for the SAM output
|
|
|
3037 }
|
|
|
3038 elsif($cigar_2 =~ tr/[NSHPX=]//){ # if these (for standard mapping) illegal characters exist we die
|
|
|
3039 die "The CIGAR 2 string contained illegal CIGAR operations in addition to 'M', 'I' and 'D': $cigar_2\n";
|
|
|
3040 }
|
|
|
3041 else{
|
|
|
3042 die "The CIGAR 2 string contained undefined CIGAR operations in addition to 'M', 'I' and 'D': $cigar_2\n";
|
|
|
3043 }
|
|
|
3044 }
|
|
|
3045
|
|
|
3046 ### 3' end of read 2
|
|
|
3047 if ( ($methylation_call_params->{$sequence_identifier}->{index} == 0) or ($methylation_call_params->{$sequence_identifier}->{index} == 2) ){
|
|
|
3048 ## checking if the substring will be valid or if we can't extract the sequence because we are right at the edge of a chromosome
|
|
|
3049 unless (length($chromosomes{$methylation_call_params->{$sequence_identifier}->{chromosome}}) >= $pos_2+2){# exiting with en empty genomic sequence otherwise
|
|
|
3050 $methylation_call_params->{$sequence_identifier}->{unmodified_genomic_sequence_2} = $non_bisulfite_sequence_2;
|
|
|
3051 return;
|
|
|
3052 }
|
|
|
3053 $non_bisulfite_sequence_2 .= substr ($chromosomes{$methylation_call_params->{$sequence_identifier}->{chromosome}},$pos_2,2);
|
|
|
3054 }
|
|
|
3055
|
|
|
3056 ### all paired-end alignments reported by Bowtie 2 have the Read 1 alignment first and the Read 2 alignment as the second one irrespective of whether read 1 or read 2 was
|
|
|
3057 ### the + alignment. We also read in sequences read 1 then read 2 so they should correspond perfectly
|
|
|
3058
|
|
|
3059 ### results from CT converted read 1 plus GA converted read 2 vs. CT converted genome (+/- orientation alignments are reported only)
|
|
|
3060 if ($methylation_call_params->{$sequence_identifier}->{index} == 0){
|
|
|
3061 ### [Index 0, sequence originated from (converted) forward strand]
|
|
|
3062 $counting{CT_GA_CT_count}++;
|
|
|
3063 $alignment_read_1 = '+';
|
|
|
3064 $alignment_read_2 = '-';
|
|
|
3065 $read_conversion_info_1 = 'CT';
|
|
|
3066 $read_conversion_info_2 = 'GA';
|
|
|
3067 $genome_conversion = 'CT';
|
|
|
3068 ### Read 1 is always the forward hit
|
|
|
3069 ### Read 2 is will always on the reverse strand, so it needs to be reverse complemented
|
|
|
3070 $non_bisulfite_sequence_2 = reverse_complement($non_bisulfite_sequence_2);
|
|
|
3071 }
|
|
|
3072
|
|
|
3073 ### results from GA converted read 1 plus CT converted read 2 vs. GA converted genome (+/- orientation alignments are reported only)
|
|
|
3074 elsif ($methylation_call_params->{$sequence_identifier}->{index} == 1){
|
|
|
3075 ### [Index 1, sequence originated from complementary to (converted) bottom strand]
|
|
|
3076 $counting{GA_CT_GA_count}++;
|
|
|
3077 $alignment_read_1 = '+';
|
|
|
3078 $alignment_read_2 = '-';
|
|
|
3079 $read_conversion_info_1 = 'GA';
|
|
|
3080 $read_conversion_info_2 = 'CT';
|
|
|
3081 $genome_conversion = 'GA';
|
|
|
3082 ### Read 1 is always the forward hit
|
|
|
3083 ### Read 2 is will always on the reverse strand, so it needs to be reverse complemented
|
|
|
3084 $non_bisulfite_sequence_2 = reverse_complement($non_bisulfite_sequence_2);
|
|
|
3085 }
|
|
|
3086
|
|
|
3087 ### results from GA converted read 1 plus CT converted read 2 vs. CT converted genome (-/+ orientation alignments are reported only)
|
|
|
3088 elsif ($methylation_call_params->{$sequence_identifier}->{index} == 2){
|
|
|
3089 ### [Index 2, sequence originated from the complementary to (converted) top strand]
|
|
|
3090 $counting{GA_CT_CT_count}++;
|
|
|
3091 $alignment_read_1 = '-';
|
|
|
3092 $alignment_read_2 = '+';
|
|
|
3093 $read_conversion_info_1 = 'GA';
|
|
|
3094 $read_conversion_info_2 = 'CT';
|
|
|
3095 $genome_conversion = 'CT';
|
|
|
3096
|
|
|
3097 ### Read 1 (the reverse strand) genomic sequence needs to be reverse complemented
|
|
|
3098 $non_bisulfite_sequence_1 = reverse_complement($non_bisulfite_sequence_1);
|
|
|
3099 }
|
|
|
3100
|
|
|
3101 ### results from CT converted read 1 plus GA converted read 2 vs. GA converted genome (-/+ orientation alignments are reported only)
|
|
|
3102 elsif ($methylation_call_params->{$sequence_identifier}->{index} == 3){
|
|
|
3103 ### [Index 3, sequence originated from the (converted) reverse strand]
|
|
|
3104 $counting{CT_GA_GA_count}++;
|
|
|
3105 $alignment_read_1 = '-';
|
|
|
3106 $alignment_read_2 = '+';
|
|
|
3107 $read_conversion_info_1 = 'CT';
|
|
|
3108 $read_conversion_info_2 = 'GA';
|
|
|
3109 $genome_conversion = 'GA';
|
|
|
3110 ### Read 1 (the reverse strand) genomic sequence needs to be reverse complemented
|
|
|
3111 $non_bisulfite_sequence_1 = reverse_complement($non_bisulfite_sequence_1);
|
|
|
3112 }
|
|
|
3113 else{
|
|
|
3114 die "Too many bowtie result filehandles\n";
|
|
|
3115 }
|
|
|
3116 ### the alignment_strand information is needed to determine which strand of the genomic sequence we are comparing the read against,
|
|
|
3117 ### the read_conversion information is needed to know whether we are looking for C->T or G->A substitutions
|
|
|
3118
|
|
|
3119 $methylation_call_params->{$sequence_identifier}->{alignment_read_1} = $alignment_read_1;
|
|
|
3120 $methylation_call_params->{$sequence_identifier}->{alignment_read_2} = $alignment_read_2;
|
|
|
3121 $methylation_call_params->{$sequence_identifier}->{genome_conversion} = $genome_conversion;
|
|
|
3122 $methylation_call_params->{$sequence_identifier}->{read_conversion_1} = $read_conversion_info_1;
|
|
|
3123 $methylation_call_params->{$sequence_identifier}->{read_conversion_2} = $read_conversion_info_2;
|
|
|
3124 $methylation_call_params->{$sequence_identifier}->{unmodified_genomic_sequence_1} = $non_bisulfite_sequence_1;
|
|
|
3125 $methylation_call_params->{$sequence_identifier}->{unmodified_genomic_sequence_2} = $non_bisulfite_sequence_2;
|
|
|
3126 ## the end position of a read is stored in $pos
|
|
|
3127 $methylation_call_params->{$sequence_identifier}->{end_position_1} = $pos_1;
|
|
|
3128 $methylation_call_params->{$sequence_identifier}->{end_position_2} = $pos_2;
|
|
|
3129 $methylation_call_params->{$sequence_identifier}->{indels_1} = $indels_1;
|
|
|
3130 $methylation_call_params->{$sequence_identifier}->{indels_2} = $indels_2;
|
|
|
3131 }
|
|
|
3132
|
|
|
3133 ##########################################
|
|
|
3134 ### PRINT SINGLE END RESULTS: Bowtie 1 ###
|
|
|
3135 ##########################################
|
|
|
3136
|
|
|
3137 sub print_bisulfite_mapping_result_single_end{
|
|
|
3138 my ($identifier,$sequence,$methylation_call_params,$quality_value)= @_;
|
|
|
3139
|
|
|
3140 ### we will output the FastQ quality in Sanger encoding (Phred 33 scale)
|
|
|
3141 if ($phred64){
|
|
|
3142 $quality_value = convert_phred64_quals_to_phred33($quality_value);
|
|
|
3143 }
|
|
|
3144 elsif ($solexa){
|
|
|
3145 $quality_value = convert_solexa_quals_to_phred33($quality_value);
|
|
|
3146 }
|
|
|
3147
|
|
|
3148 ### We will add +1 bp to the starting position of single-end reads, as Bowtie 1 reports the index and not the bp position.
|
|
|
3149 $methylation_call_params->{$identifier}->{position} += 1;
|
|
|
3150
|
|
|
3151 ### writing every uniquely mapped read and its methylation call to the output file
|
|
|
3152 if ($vanilla){
|
|
|
3153 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);
|
|
|
3154 print OUT "$bowtie1_output\n";
|
|
|
3155 }
|
|
|
3156 else{ # SAM output, default since Bismark v1.0.0
|
|
|
3157 single_end_SAM_output($identifier,$sequence,$methylation_call_params,$quality_value); # at the end of the script
|
|
|
3158 }
|
|
|
3159 }
|
|
|
3160
|
|
|
3161 ##########################################
|
|
|
3162 ### PRINT SINGLE END RESULTS: Bowtie 2 ###
|
|
|
3163 ##########################################
|
|
|
3164
|
|
|
3165 sub print_bisulfite_mapping_result_single_end_bowtie2{
|
|
|
3166 my ($identifier,$sequence,$methylation_call_params,$quality_value)= @_;
|
|
|
3167
|
|
|
3168 ### we will output the FastQ quality in Sanger encoding (Phred 33 scale)
|
|
|
3169 if ($phred64){
|
|
|
3170 $quality_value = convert_phred64_quals_to_phred33($quality_value);
|
|
|
3171 }
|
|
|
3172 elsif ($solexa){
|
|
|
3173 $quality_value = convert_solexa_quals_to_phred33($quality_value);
|
|
|
3174 }
|
|
|
3175
|
|
|
3176 ### writing every mapped read and its methylation call to the SAM output file (unmapped and ambiguous reads were already printed)
|
|
|
3177 single_end_SAM_output($identifier,$sequence,$methylation_call_params,$quality_value); # at the end of the script
|
|
|
3178 }
|
|
|
3179
|
|
|
3180 ##########################################
|
|
|
3181 ### PRINT PAIRED END ESULTS: Bowtie 1 ###
|
|
|
3182 ##########################################
|
|
|
3183
|
|
|
3184 sub print_bisulfite_mapping_results_paired_ends{
|
|
|
3185 my ($identifier,$sequence_1,$sequence_2,$methylation_call_params,$quality_value_1,$quality_value_2)= @_;
|
|
|
3186
|
|
|
3187 ### we will output the FastQ quality in Sanger encoding (Phred 33 scale)
|
|
|
3188 if ($phred64){
|
|
|
3189 $quality_value_1 = convert_phred64_quals_to_phred33($quality_value_1);
|
|
|
3190 $quality_value_2 = convert_phred64_quals_to_phred33($quality_value_2);
|
|
|
3191 }
|
|
|
3192 elsif ($solexa){
|
|
|
3193 $quality_value_1 = convert_solexa_quals_to_phred33($quality_value_1);
|
|
|
3194 $quality_value_2 = convert_solexa_quals_to_phred33($quality_value_2);
|
|
|
3195 }
|
|
|
3196
|
|
|
3197 ### 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)
|
|
|
3198 $methylation_call_params->{$identifier}->{start_seq_1} += 1;
|
|
|
3199
|
|
|
3200 ### writing every single aligned read and its methylation call to the output file
|
|
|
3201 if ($vanilla){
|
|
|
3202 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);
|
|
|
3203 print OUT "$bowtie1_output_paired_end\n";
|
|
|
3204 }
|
|
|
3205 else{ # SAM output, default since Bismark v1.0.0
|
|
|
3206 paired_end_SAM_output($identifier,$sequence_1,$sequence_2,$methylation_call_params,$quality_value_1,$quality_value_2); # at the end of the script
|
|
|
3207 }
|
|
|
3208
|
|
|
3209 }
|
|
|
3210
|
|
|
3211 ##########################################
|
|
|
3212 ### PRINT PAIRED END ESULTS: Bowtie 2 ###
|
|
|
3213 ##########################################
|
|
|
3214
|
|
|
3215 sub print_bisulfite_mapping_results_paired_ends_bowtie2{
|
|
|
3216 my ($identifier,$sequence_1,$sequence_2,$methylation_call_params,$quality_value_1,$quality_value_2)= @_;
|
|
|
3217
|
|
|
3218 ### we will output the FastQ quality in Sanger encoding (Phred 33 scale)
|
|
|
3219 if ($phred64){
|
|
|
3220 $quality_value_1 = convert_phred64_quals_to_phred33($quality_value_1);
|
|
|
3221 $quality_value_2 = convert_phred64_quals_to_phred33($quality_value_2);
|
|
|
3222 }
|
|
|
3223 elsif ($solexa){
|
|
|
3224 $quality_value_1 = convert_solexa_quals_to_phred33($quality_value_1);
|
|
|
3225 $quality_value_2 = convert_solexa_quals_to_phred33($quality_value_2);
|
|
|
3226 }
|
|
|
3227
|
|
|
3228 ### writing every single aligned read and its methylation call to the output file (unmapped and ambiguous reads were already printed)
|
|
|
3229 paired_end_SAM_output($identifier,$sequence_1,$sequence_2,$methylation_call_params,$quality_value_1,$quality_value_2); # at the end of the script
|
|
|
3230
|
|
|
3231 }
|
|
|
3232
|
|
|
3233
|
|
|
3234 sub convert_phred64_quals_to_phred33{
|
|
|
3235
|
|
|
3236 my $qual = shift;
|
|
|
3237 my @quals = split (//,$qual);
|
|
|
3238 my @new_quals;
|
|
|
3239
|
|
|
3240 foreach my $index (0..$#quals){
|
|
|
3241 my $phred_score = convert_phred64_quality_string_into_phred_score ($quals[$index]);
|
|
|
3242 my $phred33_quality_string = convert_phred_score_into_phred33_quality_string ($phred_score);
|
|
|
3243 $new_quals[$index] = $phred33_quality_string;
|
|
|
3244 }
|
|
|
3245
|
|
|
3246 my $phred33_quality = join ("",@new_quals);
|
|
|
3247 return $phred33_quality;
|
|
|
3248 }
|
|
|
3249
|
|
|
3250 sub convert_solexa_quals_to_phred33{
|
|
|
3251
|
|
|
3252 my $qual = shift;
|
|
|
3253 my @quals = split (//,$qual);
|
|
|
3254 my @new_quals;
|
|
|
3255
|
|
|
3256 foreach my $index (0..$#quals){
|
|
|
3257 my $phred_score = convert_solexa_pre1_3_quality_string_into_phred_score ($quals[$index]);
|
|
|
3258 my $phred33_quality_string = convert_phred_score_into_phred33_quality_string ($phred_score);
|
|
|
3259 $new_quals[$index] = $phred33_quality_string;
|
|
|
3260 }
|
|
|
3261
|
|
|
3262 my $phred33_quality = join ("",@new_quals);
|
|
|
3263 return $phred33_quality;
|
|
|
3264 }
|
|
|
3265
|
|
|
3266 sub convert_phred_score_into_phred33_quality_string{
|
|
|
3267 my $qual = shift;
|
|
|
3268 $qual = chr($qual+33);
|
|
|
3269 return $qual;
|
|
|
3270 }
|
|
|
3271
|
|
|
3272 sub convert_phred64_quality_string_into_phred_score{
|
|
|
3273 my $string = shift;
|
|
|
3274 my $qual = ord($string)-64;
|
|
|
3275 return $qual;
|
|
|
3276 }
|
|
|
3277
|
|
|
3278 sub convert_solexa_pre1_3_quality_string_into_phred_score{
|
|
|
3279 ### 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
|
|
|
3280 my $string = shift;
|
|
|
3281 my $qual = ord($string)-59;
|
|
|
3282 return $qual;
|
|
|
3283 }
|
|
|
3284
|
|
|
3285
|
|
|
3286 sub extract_corresponding_genomic_sequence_single_end {
|
|
|
3287 my ($sequence_identifier,$methylation_call_params) = @_;
|
|
|
3288 ### A bisulfite sequence for 1 location in the genome can theoretically be any of the 4 possible converted strands. We are also giving the
|
|
|
3289 ### sequence a 'memory' of the conversion we are expecting which we will need later for the methylation call
|
|
|
3290
|
|
|
3291 ### the alignment_strand information is needed to determine which strand of the genomic sequence we are comparing the read against,
|
|
|
3292 ### the read_conversion information is needed to know whether we are looking for C->T or G->A substitutions
|
|
|
3293 my $alignment_strand;
|
|
|
3294 my $read_conversion_info;
|
|
|
3295 my $genome_conversion;
|
|
|
3296 ### Also extracting the corresponding genomic sequence, +2 extra bases at the end so that we can also make a CpG methylation call and
|
|
|
3297 ### in addition make differential calls for Cs non-CpG context, which will now be divided into CHG and CHH methylation,
|
|
|
3298 ### if the C happens to be at the last position of the actually observed sequence
|
|
|
3299 my $non_bisulfite_sequence;
|
|
|
3300 ### depending on the conversion we want to make need to capture 1 extra base at the 3' end
|
|
|
3301
|
|
|
3302 ### results from CT converted read vs. CT converted genome (+ orientation alignments are reported only)
|
|
|
3303 if ($methylation_call_params->{$sequence_identifier}->{index} == 0){
|
|
|
3304 ### [Index 0, sequence originated from (converted) forward strand]
|
|
|
3305 $counting{CT_CT_count}++;
|
|
|
3306 $alignment_strand = '+';
|
|
|
3307 $read_conversion_info = 'CT';
|
|
|
3308 $genome_conversion = 'CT';
|
|
|
3309
|
|
|
3310 ## checking if the substring will be valid or if we can't extract the sequence because we are right at the edge of a chromosome
|
|
|
3311 if (length($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
|
|
|
3312 ### + 2 extra base at the 3' end
|
|
|
3313 $non_bisulfite_sequence = substr ($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
|
|
|
3314 }
|
|
|
3315 else{
|
|
|
3316 $non_bisulfite_sequence = '';
|
|
|
3317 }
|
|
|
3318 }
|
|
|
3319
|
|
|
3320 ### results from CT converted reads vs. GA converted genome (- orientation alignments are reported only)
|
|
|
3321 elsif ($methylation_call_params->{$sequence_identifier}->{index} == 1){
|
|
|
3322 ### [Index 1, sequence originated from (converted) reverse strand]
|
|
|
3323 $counting{CT_GA_count}++;
|
|
|
3324 $alignment_strand = '-';
|
|
|
3325 $read_conversion_info = 'CT';
|
|
|
3326 $genome_conversion = 'GA';
|
|
|
3327
|
|
|
3328 ## checking if the substring will be valid or if we can't extract the sequence because we are right at the edge of a chromosome
|
|
|
3329 if ($methylation_call_params->{$sequence_identifier}->{position}-2 >= 0){ ## CHH changed to -2 # 02 02 2012 Changed this to >= from >
|
|
|
3330 ### Extracting 2 extra 5' bases on forward strand which will become 2 extra 3' bases after reverse complementation
|
|
|
3331 $non_bisulfite_sequence = substr ($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
|
|
|
3332 ## reverse complement!
|
|
|
3333 $non_bisulfite_sequence = reverse_complement($non_bisulfite_sequence);
|
|
|
3334 }
|
|
|
3335 else{
|
|
|
3336 $non_bisulfite_sequence = '';
|
|
|
3337 }
|
|
|
3338 }
|
|
|
3339
|
|
|
3340 ### results from GA converted reads vs. CT converted genome (- orientation alignments are reported only)
|
|
|
3341 elsif ($methylation_call_params->{$sequence_identifier}->{index} == 2){
|
|
|
3342 ### [Index 2, sequence originated from complementary to (converted) forward strand]
|
|
|
3343 $counting{GA_CT_count}++;
|
|
|
3344 $alignment_strand = '-';
|
|
|
3345 $read_conversion_info = 'GA';
|
|
|
3346 $genome_conversion = 'CT';
|
|
|
3347
|
|
|
3348 ### +2 extra bases on the forward strand 3', which will become 2 extra 5' bases after reverse complementation
|
|
|
3349 ## checking if the substring will be valid or if we can't extract the sequence because we are right at the edge of a chromosome
|
|
|
3350 if (length($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
|
|
|
3351 $non_bisulfite_sequence = substr ($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
|
|
|
3352 ## reverse complement!
|
|
|
3353 $non_bisulfite_sequence = reverse_complement($non_bisulfite_sequence);
|
|
|
3354 }
|
|
|
3355 else{
|
|
|
3356 $non_bisulfite_sequence = '';
|
|
|
3357 }
|
|
|
3358 }
|
|
|
3359
|
|
|
3360 ### results from GA converted reads vs. GA converted genome (+ orientation alignments are reported only)
|
|
|
3361 elsif ($methylation_call_params->{$sequence_identifier}->{index} == 3){
|
|
|
3362 ### [Index 3, sequence originated from complementary to (converted) reverse strand]
|
|
|
3363 $counting{GA_GA_count}++;
|
|
|
3364 $alignment_strand = '+';
|
|
|
3365 $read_conversion_info = 'GA';
|
|
|
3366 $genome_conversion = 'GA';
|
|
|
3367
|
|
|
3368 ## checking if the substring will be valid or if we can't extract the sequence because we are right at the edge of a chromosome
|
|
|
3369 if ($methylation_call_params->{$sequence_identifier}->{position}-2 >= 0){ ## CHH changed to +2 # 02 02 2012 Changed this to >= from >
|
|
|
3370 ### +2 extra base at the 5' end as we are nominally checking the converted reverse strand
|
|
|
3371 $non_bisulfite_sequence = substr ($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
|
|
|
3372 }
|
|
|
3373 else{
|
|
|
3374 $non_bisulfite_sequence = '';
|
|
|
3375 }
|
|
|
3376 }
|
|
|
3377 else{
|
|
|
3378 die "Too many bowtie result filehandles\n";
|
|
|
3379 }
|
|
|
3380
|
|
|
3381 $methylation_call_params->{$sequence_identifier}->{alignment_strand} = $alignment_strand;
|
|
|
3382 $methylation_call_params->{$sequence_identifier}->{read_conversion} = $read_conversion_info;
|
|
|
3383 $methylation_call_params->{$sequence_identifier}->{genome_conversion} = $genome_conversion;
|
|
|
3384 $methylation_call_params->{$sequence_identifier}->{unmodified_genomic_sequence} = $non_bisulfite_sequence;
|
|
|
3385
|
|
|
3386 ### at this point we can also determine the end position of a read
|
|
|
3387 $methylation_call_params->{$sequence_identifier}->{end_position} = $methylation_call_params->{$sequence_identifier}->{position}+length($methylation_call_params->{$sequence_identifier}->{bowtie_sequence});
|
|
|
3388 }
|
|
|
3389
|
|
|
3390
|
|
|
3391 sub extract_corresponding_genomic_sequence_single_end_bowtie2{
|
|
|
3392 my ($sequence_identifier,$methylation_call_params) = @_;
|
|
|
3393
|
|
|
3394 my $MD_tag = $methylation_call_params->{$sequence_identifier}->{mismatch_info};
|
|
|
3395 my $cigar = $methylation_call_params->{$sequence_identifier}->{CIGAR};
|
|
|
3396
|
|
|
3397 ### A bisulfite sequence for 1 location in the genome can theoretically be any of the 4 possible converted strands. We are also giving the
|
|
|
3398 ### sequence a 'memory' of the conversion we are expecting which we will need later for the methylation call
|
|
|
3399
|
|
|
3400 ### the alignment_strand information is needed to determine which strand of the genomic sequence we are comparing the read against,
|
|
|
3401 ### the read_conversion information is needed to know whether we are looking for C->T or G->A substitutions
|
|
|
3402 my $alignment_strand;
|
|
|
3403 my $read_conversion_info;
|
|
|
3404 my $genome_conversion;
|
|
|
3405 ### 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
|
|
|
3406 ### 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
|
|
|
3407 my $non_bisulfite_sequence = '';
|
|
|
3408
|
|
|
3409 ### Positions in SAM format are 1 based, so we need to subract 1 when getting substrings
|
|
|
3410 my $pos = $methylation_call_params->{$sequence_identifier}->{position}-1;
|
|
|
3411
|
|
|
3412 # parsing CIGAR string
|
|
|
3413 my @len = split (/\D+/,$cigar); # storing the length per operation
|
|
|
3414 my @ops = split (/\d+/,$cigar); # storing the operation
|
|
|
3415 shift @ops; # remove the empty first element
|
|
|
3416 die "CIGAR string contained a non-matching number of lengths and operations\n" unless (scalar @len == scalar @ops);
|
|
|
3417
|
|
|
3418 ### 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)
|
|
|
3419 if ( ($methylation_call_params->{$sequence_identifier}->{index} == 1) or ($methylation_call_params->{$sequence_identifier}->{index} == 3) ){
|
|
|
3420 ## 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
|
|
|
3421 unless ( ($pos-2) >= 0){ # exiting with en empty genomic sequence otherwise
|
|
|
3422 $methylation_call_params->{$sequence_identifier}->{unmodified_genomic_sequence} = $non_bisulfite_sequence;
|
|
|
3423 return;
|
|
|
3424 }
|
|
|
3425 $non_bisulfite_sequence .= substr ($chromosomes{$methylation_call_params->{$sequence_identifier}->{chromosome}},$pos-2,2);
|
|
|
3426 }
|
|
|
3427 my $indels = 0;
|
|
|
3428
|
|
|
3429 foreach (0..$#len){
|
|
|
3430 if ($ops[$_] eq 'M'){
|
|
|
3431 #extracting genomic sequence
|
|
|
3432 $non_bisulfite_sequence .= substr ($chromosomes{$methylation_call_params->{$sequence_identifier}->{chromosome}},$pos,$len[$_]);
|
|
|
3433 # adjusting position
|
|
|
3434 $pos += $len[$_];
|
|
|
3435 }
|
|
|
3436 elsif ($ops[$_] eq 'I'){ # insertion in the read sequence
|
|
|
3437 # we simply add padding Ns instead of finding genomic sequence. This will not be used to infer methylation calls
|
|
|
3438 $non_bisulfite_sequence .= 'N' x $len[$_];
|
|
|
3439 # warn "$non_bisulfite_sequence\n";
|
|
|
3440 # position doesn't need to be adjusting
|
|
|
3441 $indels += $len[$_]; # adding this to $indels so we can determine the hemming distance for the SAM output (= single-base substitutions (mismatches, insertions, deletions)
|
|
|
3442 }
|
|
|
3443 elsif ($ops[$_] eq 'D'){ # deletion in the read sequence
|
|
|
3444 # we do not add any genomic sequence but only adjust the position
|
|
|
3445 $pos += $len[$_];
|
|
|
3446 $indels += $len[$_]; # adding this to $indels so we can determine the hemming distance for the SAM output (= single-base substitutions (mismatches, insertions, deletions)
|
|
|
3447 }
|
|
|
3448 elsif($cigar =~ tr/[NSHPX=]//){ # if these (for standard mapping) illegal characters exist we die
|
|
|
3449 die "The CIGAR string contained illegal CIGAR operations in addition to 'M', 'I' and 'D': $cigar\n";
|
|
|
3450 }
|
|
|
3451 else{
|
|
|
3452 die "The CIGAR string contained undefined CIGAR operations in addition to 'M', 'I' and 'D': $cigar\n";
|
|
|
3453 }
|
|
|
3454 }
|
|
|
3455
|
|
|
3456 ### If the sequence aligns best as CT converted reads vs. CT converted genome (OT, index 0) or GA converted reads vs. CT converted genome (CTOT, index 2)
|
|
|
3457 if ( ($methylation_call_params->{$sequence_identifier}->{index} == 0) or ($methylation_call_params->{$sequence_identifier}->{index} == 2) ){
|
|
|
3458 ## checking if the substring will be valid or if we can't extract the sequence because we are right at the edge of a chromosome
|
|
|
3459 unless (length($chromosomes{$methylation_call_params->{$sequence_identifier}->{chromosome}}) >= $pos+2){ # exiting with en empty genomic sequence otherwise
|
|
|
3460 $methylation_call_params->{$sequence_identifier}->{unmodified_genomic_sequence} = $non_bisulfite_sequence;
|
|
|
3461 return;
|
|
|
3462 }
|
|
|
3463 $non_bisulfite_sequence .= substr ($chromosomes{$methylation_call_params->{$sequence_identifier}->{chromosome}},$pos,2);
|
|
|
3464 # print "$methylation_call_params->{$sequence_identifier}->{bowtie_sequence}\n$non_bisulfite_sequence\n";
|
|
|
3465 }
|
|
|
3466
|
|
|
3467
|
|
|
3468
|
|
|
3469 ### results from CT converted read vs. CT converted genome (+ orientation alignments are reported only)
|
|
|
3470 if ($methylation_call_params->{$sequence_identifier}->{index} == 0){
|
|
|
3471 ### [Index 0, sequence originated from (converted) forward strand]
|
|
|
3472 $counting{CT_CT_count}++;
|
|
|
3473 $alignment_strand = '+';
|
|
|
3474 $read_conversion_info = 'CT';
|
|
|
3475 $genome_conversion = 'CT';
|
|
|
3476 }
|
|
|
3477
|
|
|
3478 ### results from CT converted reads vs. GA converted genome (- orientation alignments are reported only)
|
|
|
3479 elsif ($methylation_call_params->{$sequence_identifier}->{index} == 1){
|
|
|
3480 ### [Index 1, sequence originated from (converted) reverse strand]
|
|
|
3481 $counting{CT_GA_count}++;
|
|
|
3482 $alignment_strand = '-';
|
|
|
3483 $read_conversion_info = 'CT';
|
|
|
3484 $genome_conversion = 'GA';
|
|
|
3485
|
|
|
3486 ### reverse complement!
|
|
|
3487 $non_bisulfite_sequence = reverse_complement($non_bisulfite_sequence);
|
|
|
3488 }
|
|
|
3489
|
|
|
3490 ### results from GA converted reads vs. CT converted genome (- orientation alignments are reported only)
|
|
|
3491 elsif ($methylation_call_params->{$sequence_identifier}->{index} == 2){
|
|
|
3492 ### [Index 2, sequence originated from complementary to (converted) forward strand]
|
|
|
3493 $counting{GA_CT_count}++;
|
|
|
3494 $alignment_strand = '-';
|
|
|
3495 $read_conversion_info = 'GA';
|
|
|
3496 $genome_conversion = 'CT';
|
|
|
3497
|
|
|
3498 ### reverse complement!
|
|
|
3499 $non_bisulfite_sequence = reverse_complement($non_bisulfite_sequence);
|
|
|
3500 }
|
|
|
3501
|
|
|
3502 ### results from GA converted reads vs. GA converted genome (+ orientation alignments are reported only)
|
|
|
3503 elsif ($methylation_call_params->{$sequence_identifier}->{index} == 3){
|
|
|
3504 ### [Index 3, sequence originated from complementary to (converted) reverse strand]
|
|
|
3505 $counting{GA_GA_count}++;
|
|
|
3506 $alignment_strand = '+';
|
|
|
3507 $read_conversion_info = 'GA';
|
|
|
3508 $genome_conversion = 'GA';
|
|
|
3509
|
|
|
3510 }
|
|
|
3511 else{
|
|
|
3512 die "Too many Bowtie 2 result filehandles\n";
|
|
|
3513 }
|
|
|
3514
|
|
|
3515 $methylation_call_params->{$sequence_identifier}->{alignment_strand} = $alignment_strand;
|
|
|
3516 $methylation_call_params->{$sequence_identifier}->{read_conversion} = $read_conversion_info;
|
|
|
3517 $methylation_call_params->{$sequence_identifier}->{genome_conversion} = $genome_conversion;
|
|
|
3518 $methylation_call_params->{$sequence_identifier}->{unmodified_genomic_sequence} = $non_bisulfite_sequence;
|
|
|
3519
|
|
|
3520 ### the end position of a read is stored in $pos
|
|
|
3521 $methylation_call_params->{$sequence_identifier}->{end_position} = $pos;
|
|
|
3522 $methylation_call_params->{$sequence_identifier}->{indels} = $indels;
|
|
|
3523 }
|
|
|
3524
|
|
|
3525 ### METHYLATION CALL
|
|
|
3526
|
|
|
3527 sub methylation_call{
|
|
|
3528 my ($identifier,$sequence_actually_observed,$genomic_sequence,$read_conversion) = @_;
|
|
|
3529 ### splitting both the actually observed sequence and the genomic sequence up into single bases so we can compare them one by one
|
|
|
3530 my @seq = split(//,$sequence_actually_observed);
|
|
|
3531 my @genomic = split(//,$genomic_sequence);
|
|
|
3532 # print join ("\n",$identifier,$sequence_actually_observed,$genomic_sequence,$read_conversion),"\n";
|
|
|
3533 ### Creating a match-string with different characters for non-cytosine bases (disregarding mismatches here), methyl-Cs or non-methyl Cs in either
|
|
|
3534 ### CpG, CHH or CHG context
|
|
|
3535
|
|
|
3536 #################################################################
|
|
|
3537 ### . for bases not involving cytosines ###
|
|
|
3538 ### X for methylated C in CHG context (was protected) ###
|
|
|
3539 ### x for not methylated C in CHG context (was converted) ###
|
|
|
3540 ### H for methylated C in CHH context (was protected) ###
|
|
|
3541 ### h for not methylated C in CHH context (was converted) ###
|
|
|
3542 ### Z for methylated C in CpG context (was protected) ###
|
|
|
3543 ### z for not methylated C in CpG context (was converted) ###
|
|
|
3544 #################################################################
|
|
|
3545
|
|
|
3546 my @match =();
|
|
|
3547 warn "length of \@seq: ",scalar @seq,"\tlength of \@genomic: ",scalar @genomic,"\n" unless (scalar @seq eq (scalar@genomic-2)); ## CHH changed to -2
|
|
|
3548 my $methyl_CHH_count = 0;
|
|
|
3549 my $methyl_CHG_count = 0;
|
|
|
3550 my $methyl_CpG_count = 0;
|
|
|
3551 my $unmethylated_CHH_count = 0;
|
|
|
3552 my $unmethylated_CHG_count = 0;
|
|
|
3553 my $unmethylated_CpG_count = 0;
|
|
|
3554
|
|
|
3555 if ($read_conversion eq 'CT'){
|
|
|
3556 for my $index (0..$#seq) {
|
|
|
3557 if ($seq[$index] eq $genomic[$index]) {
|
|
|
3558 ### The residue can only be a C if it was not converted to T, i.e. protected my methylation
|
|
|
3559 if ($genomic[$index] eq 'C') {
|
|
|
3560 ### If the residue is a C we want to know if it was in CpG context or in any other context
|
|
|
3561 my $downstream_base = $genomic[$index+1];
|
|
|
3562
|
|
|
3563 if ($downstream_base eq 'G'){
|
|
|
3564 ++$methyl_CpG_count;
|
|
|
3565 push @match,'Z'; # protected C, methylated, in CpG context
|
|
|
3566 }
|
|
|
3567
|
|
|
3568 else {
|
|
|
3569 ### C in not in CpG-context, determining the second downstream base context
|
|
|
3570 my $second_downstream_base = $genomic[$index+2];
|
|
|
3571
|
|
|
3572 if ($second_downstream_base eq 'G'){
|
|
|
3573 ++$methyl_CHG_count;
|
|
|
3574 push @match,'X'; # protected C, methylated, in CHG context
|
|
|
3575 }
|
|
|
3576 else{
|
|
|
3577 ++$methyl_CHH_count;
|
|
|
3578 push @match,'H'; # protected C, methylated, in CHH context
|
|
|
3579 }
|
|
|
3580 }
|
|
|
3581 }
|
|
|
3582 else {
|
|
|
3583 push @match, '.';
|
|
|
3584 }
|
|
|
3585 }
|
|
|
3586 elsif ($seq[$index] ne $genomic[$index]) {
|
|
|
3587 ### for the methylation call we are only interested in mismatches involving cytosines (in the genomic sequence) which were converted into Ts
|
|
|
3588 ### in the actually observed sequence
|
|
|
3589 if ($genomic[$index] eq 'C' and $seq[$index] eq 'T') {
|
|
|
3590 ### If the residue was converted to T we want to know if it was in CpG, CHG or CHH context
|
|
|
3591 my $downstream_base = $genomic[$index+1];
|
|
|
3592
|
|
|
3593 if ($downstream_base eq 'G'){
|
|
|
3594 ++$unmethylated_CpG_count;
|
|
|
3595 push @match,'z'; # converted C, not methylated, in CpG context
|
|
|
3596 }
|
|
|
3597
|
|
|
3598 else{
|
|
|
3599 ### C in not in CpG-context, determining the second downstream base context
|
|
|
3600 my $second_downstream_base = $genomic[$index+2];
|
|
|
3601
|
|
|
3602 if ($second_downstream_base eq 'G'){
|
|
|
3603 ++$unmethylated_CHG_count;
|
|
|
3604 push @match,'x'; # converted C, not methylated, in CHG context
|
|
|
3605 }
|
|
|
3606 else{
|
|
|
3607 ++$unmethylated_CHH_count;
|
|
|
3608 push @match,'h'; # converted C, not methylated, in CHH context
|
|
|
3609 }
|
|
|
3610 }
|
|
|
3611 }
|
|
|
3612 ### all other mismatches are not of interest for a methylation call
|
|
|
3613 else {
|
|
|
3614 push @match,'.';
|
|
|
3615 }
|
|
|
3616 }
|
|
|
3617 else{
|
|
|
3618 die "There can be only 2 possibilities\n";
|
|
|
3619 }
|
|
|
3620 }
|
|
|
3621 }
|
|
|
3622 elsif ($read_conversion eq 'GA'){
|
|
|
3623 # print join ("\n",'***',$identifier,$sequence_actually_observed,$genomic_sequence,$read_conversion,'***'),"\n";
|
|
|
3624
|
|
|
3625 for my $index (0..$#seq) {
|
|
|
3626 if ($seq[$index] eq $genomic[$index+2]) {
|
|
|
3627 ### The residue can only be a G if the C on the other strand was not converted to T, i.e. protected my methylation
|
|
|
3628 if ($genomic[$index+2] eq 'G') {
|
|
|
3629 ### 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
|
|
|
3630 ### to look if the base upstream is a C
|
|
|
3631
|
|
|
3632 my $upstream_base = $genomic[$index+1];
|
|
|
3633
|
|
|
3634 if ($upstream_base eq 'C'){
|
|
|
3635 ++$methyl_CpG_count;
|
|
|
3636 push @match,'Z'; # protected C on opposing strand, methylated, in CpG context
|
|
|
3637 }
|
|
|
3638
|
|
|
3639 else{
|
|
|
3640 ### C in not in CpG-context, determining the second upstream base context
|
|
|
3641 my $second_upstream_base = $genomic[$index];
|
|
|
3642
|
|
|
3643 if ($second_upstream_base eq 'C'){
|
|
|
3644 ++$methyl_CHG_count;
|
|
|
3645 push @match,'X'; # protected C on opposing strand, methylated, in CHG context
|
|
|
3646 }
|
|
|
3647 else{
|
|
|
3648 ++$methyl_CHH_count;
|
|
|
3649 push @match,'H'; # protected C on opposing strand, methylated, in CHH context
|
|
|
3650 }
|
|
|
3651 }
|
|
|
3652 }
|
|
|
3653 else{
|
|
|
3654 push @match, '.';
|
|
|
3655 }
|
|
|
3656 }
|
|
|
3657 elsif ($seq[$index] ne $genomic[$index+2]) {
|
|
|
3658 ### for the methylation call we are only interested in mismatches involving cytosines (in the genomic sequence) which were converted to Ts
|
|
|
3659 ### on the opposing strand, so G to A conversions in the actually observed sequence
|
|
|
3660 if ($genomic[$index+2] eq 'G' and $seq[$index] eq 'A') {
|
|
|
3661 ### 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
|
|
|
3662 ### 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!
|
|
|
3663
|
|
|
3664 my $upstream_base = $genomic[$index+1];
|
|
|
3665
|
|
|
3666 if ($upstream_base eq 'C'){
|
|
|
3667 ++$unmethylated_CpG_count;
|
|
|
3668 push @match,'z'; # converted C on opposing strand, not methylated, in CpG context
|
|
|
3669 }
|
|
|
3670
|
|
|
3671 else{
|
|
|
3672 ### C in not in CpG-context, determining the second upstream base context
|
|
|
3673 my $second_upstream_base = $genomic[$index];
|
|
|
3674
|
|
|
3675 if ($second_upstream_base eq 'C'){
|
|
|
3676 ++$unmethylated_CHG_count;
|
|
|
3677 push @match,'x'; # converted C on opposing strand, not methylated, in CHG context
|
|
|
3678 }
|
|
|
3679 else{
|
|
|
3680 ++$unmethylated_CHH_count;
|
|
|
3681 push @match,'h'; # converted C on opposing strand, not methylated, in CHH context
|
|
|
3682 }
|
|
|
3683 }
|
|
|
3684 }
|
|
|
3685 ### all other mismatches are not of interest for a methylation call
|
|
|
3686 else {
|
|
|
3687 push @match,'.';
|
|
|
3688 }
|
|
|
3689 }
|
|
|
3690 else{
|
|
|
3691 die "There can be only 2 possibilities\n";
|
|
|
3692 }
|
|
|
3693 }
|
|
|
3694 }
|
|
|
3695 else{
|
|
|
3696 die "Strand conversion info is required to perform a methylation call\n";
|
|
|
3697 }
|
|
|
3698
|
|
|
3699 my $methylation_call = join ("",@match);
|
|
|
3700
|
|
|
3701 $counting{total_meCHH_count} += $methyl_CHH_count;
|
|
|
3702 $counting{total_meCHG_count} += $methyl_CHG_count;
|
|
|
3703 $counting{total_meCpG_count} += $methyl_CpG_count;
|
|
|
3704 $counting{total_unmethylated_CHH_count} += $unmethylated_CHH_count;
|
|
|
3705 $counting{total_unmethylated_CHG_count} += $unmethylated_CHG_count;
|
|
|
3706 $counting{total_unmethylated_CpG_count} += $unmethylated_CpG_count;
|
|
|
3707
|
|
|
3708 # print "\n$sequence_actually_observed\n$genomic_sequence\n",@match,"\n$read_conversion\n\n";
|
|
|
3709 return $methylation_call;
|
|
|
3710 }
|
|
|
3711
|
|
|
3712 sub read_genome_into_memory{
|
|
|
3713 ## working directoy
|
|
|
3714 my $cwd = shift;
|
|
|
3715 ## reading in and storing the specified genome in the %chromosomes hash
|
|
|
3716 chdir ($genome_folder) or die "Can't move to $genome_folder: $!";
|
|
|
3717 print "Now reading in and storing sequence information of the genome specified in: $genome_folder\n\n";
|
|
|
3718
|
|
|
3719 my @chromosome_filenames = <*.fa>;
|
|
|
3720
|
|
|
3721 ### if there aren't any genomic files with the extension .fa we will look for files with the extension .fasta
|
|
|
3722 unless (@chromosome_filenames){
|
|
|
3723 @chromosome_filenames = <*.fasta>;
|
|
|
3724 }
|
|
|
3725
|
|
|
3726 unless (@chromosome_filenames){
|
|
|
3727 die "The specified genome folder $genome_folder does not contain any sequence files in FastA format (with .fa or .fasta file extensions)\n";
|
|
|
3728 }
|
|
|
3729
|
|
|
3730 foreach my $chromosome_filename (@chromosome_filenames){
|
|
|
3731
|
|
|
3732 open (CHR_IN,$chromosome_filename) or die "Failed to read from sequence file $chromosome_filename $!\n";
|
|
|
3733 ### first line needs to be a fastA header
|
|
|
3734 my $first_line = <CHR_IN>;
|
|
|
3735 chomp $first_line;
|
|
|
3736
|
|
|
3737 ### Extracting chromosome name from the FastA header
|
|
|
3738 my $chromosome_name = extract_chromosome_name($first_line);
|
|
|
3739
|
|
|
3740 my $sequence;
|
|
|
3741 while (<CHR_IN>){
|
|
|
3742 chomp;
|
|
|
3743 if ($_ =~ /^>/){
|
|
|
3744 ### storing the previous chromosome in the %chromosomes hash, only relevant for Multi-Fasta-Files (MFA)
|
|
|
3745 if (exists $chromosomes{$chromosome_name}){
|
|
|
3746 print "chr $chromosome_name (",length $sequence ," bp)\n";
|
|
|
3747 die "Exiting because chromosome name already exists. Please make sure all chromosomes have a unique name!\n";
|
|
|
3748 }
|
|
|
3749 else {
|
|
|
3750 if (length($sequence) == 0){
|
|
|
3751 warn "Chromosome $chromosome_name in the multi-fasta file $chromosome_filename did not contain any sequence information!\n";
|
|
|
3752 }
|
|
|
3753 print "chr $chromosome_name (",length $sequence ," bp)\n";
|
|
|
3754 $chromosomes{$chromosome_name} = $sequence;
|
|
|
3755 }
|
|
|
3756 ### resetting the sequence variable
|
|
|
3757 $sequence = '';
|
|
|
3758 ### setting new chromosome name
|
|
|
3759 $chromosome_name = extract_chromosome_name($_);
|
|
|
3760 }
|
|
|
3761 else{
|
|
|
3762 $sequence .= uc$_;
|
|
|
3763 }
|
|
|
3764 }
|
|
|
3765
|
|
|
3766 if (exists $chromosomes{$chromosome_name}){
|
|
|
3767 print "chr $chromosome_name (",length $sequence ," bp)\t";
|
|
|
3768 die "Exiting because chromosome name already exists. Please make sure all chromosomes have a unique name.\n";
|
|
|
3769 }
|
|
|
3770 else{
|
|
|
3771 if (length($sequence) == 0){
|
|
|
3772 warn "Chromosome $chromosome_name in the file $chromosome_filename did not contain any sequence information!\n";
|
|
|
3773 }
|
|
|
3774 print "chr $chromosome_name (",length $sequence ," bp)\n";
|
|
|
3775 $chromosomes{$chromosome_name} = $sequence;
|
|
|
3776 }
|
|
|
3777 }
|
|
|
3778 print "\n";
|
|
|
3779 chdir $cwd or die "Failed to move to directory $cwd\n";
|
|
|
3780 }
|
|
|
3781
|
|
|
3782 sub extract_chromosome_name {
|
|
|
3783 ## Bowtie seems to extract the first string after the inition > in the FASTA file, so we are doing this as well
|
|
|
3784 my $fasta_header = shift;
|
|
|
3785 if ($fasta_header =~ s/^>//){
|
|
|
3786 my ($chromosome_name) = split (/\s+/,$fasta_header);
|
|
|
3787 return $chromosome_name;
|
|
|
3788 }
|
|
|
3789 else{
|
|
|
3790 die "The specified chromosome ($fasta_header) file doesn't seem to be in FASTA format as required!\n";
|
|
|
3791 }
|
|
|
3792 }
|
|
|
3793
|
|
|
3794 sub reverse_complement{
|
|
|
3795 my $sequence = shift;
|
|
|
3796 $sequence =~ tr/CATG/GTAC/;
|
|
|
3797 $sequence = reverse($sequence);
|
|
|
3798 return $sequence;
|
|
|
3799 }
|
|
|
3800
|
|
|
3801 sub biTransformFastAFiles {
|
|
|
3802 my $file = shift;
|
|
|
3803 my ($dir,$filename);
|
|
|
3804 if ($file =~ /\//){
|
|
|
3805 ($dir,$filename) = $file =~ m/(.*\/)(.*)$/;
|
|
|
3806 }
|
|
|
3807 else{
|
|
|
3808 $filename = $file;
|
|
|
3809 }
|
|
|
3810
|
|
|
3811 ### gzipped version of the infile
|
|
|
3812 if ($file =~ /\.gz$/){
|
|
|
3813 open (IN,"zcat $file |") or die "Couldn't read from file $file: $!\n";
|
|
|
3814 }
|
|
|
3815 else{
|
|
|
3816 open (IN,$file) or die "Couldn't read from file $file: $!\n";
|
|
|
3817 }
|
|
|
3818
|
|
|
3819 if ($skip){
|
|
|
3820 warn "Skipping the first $skip reads from $file\n";
|
|
|
3821 sleep (1);
|
|
|
3822 }
|
|
|
3823 if ($upto){
|
|
|
3824 warn "Processing reads up to sequence no. $upto from $file\n";
|
|
|
3825 sleep (1);
|
|
|
3826 }
|
|
|
3827
|
|
|
3828 my $C_to_T_infile = my $G_to_A_infile = $filename;
|
|
|
3829 $C_to_T_infile =~ s/$/_C_to_T.fa/;
|
|
|
3830 $G_to_A_infile =~ s/$/_G_to_A.fa/;
|
|
|
3831 print "Writing a C -> T converted version of the input file $filename to $temp_dir$C_to_T_infile\n";
|
|
|
3832 open (CTOT,'>',"$temp_dir$C_to_T_infile") or die "Couldn't write to file $!\n";
|
|
|
3833
|
|
|
3834 unless ($directional){
|
|
|
3835 print "Writing a G -> A converted version of the input file $filename to $temp_dir$G_to_A_infile\n";
|
|
|
3836 open (GTOA,'>',"$temp_dir$G_to_A_infile") or die "Couldn't write to file $!\n";
|
|
|
3837 }
|
|
|
3838
|
|
|
3839 my $count = 0;
|
|
|
3840 while (1){
|
|
|
3841 my $header = <IN>;
|
|
|
3842 my $sequence= <IN>;
|
|
|
3843 last unless ($header and $sequence);
|
|
|
3844
|
|
|
3845 $header = fix_IDs($header); # this is to avoid problems with truncated read ID when they contain white spaces
|
|
|
3846
|
|
|
3847 ++$count;
|
|
|
3848
|
|
|
3849 if ($skip){
|
|
|
3850 next unless ($count > $skip);
|
|
|
3851 }
|
|
|
3852 if ($upto){
|
|
|
3853 last if ($count > $upto);
|
|
|
3854 }
|
|
|
3855
|
|
|
3856 $sequence = uc$sequence; # make input file case insensitive
|
|
|
3857
|
|
|
3858 # detecting if the input file contains tab stops, as this is likely to result in no alignments
|
|
|
3859 if (index($header,"\t") != -1){
|
|
|
3860 $seqID_contains_tabs++;
|
|
|
3861 }
|
|
|
3862
|
|
|
3863 ### small check if the sequence seems to be in FastA format
|
|
|
3864 die "Input file doesn't seem to be in FastA format at sequence $count: $!\n" unless ($header =~ /^>.*/);
|
|
|
3865
|
|
|
3866 my $sequence_C_to_T = $sequence;
|
|
|
3867 $sequence_C_to_T =~ tr/C/T/;
|
|
|
3868 print CTOT "$header$sequence_C_to_T";
|
|
|
3869
|
|
|
3870 unless ($directional){
|
|
|
3871 my $sequence_G_to_A = $sequence;
|
|
|
3872 $sequence_G_to_A =~ tr/G/A/;
|
|
|
3873 print GTOA "$header$sequence_G_to_A";
|
|
|
3874 }
|
|
|
3875 }
|
|
|
3876 if ($directional){
|
|
|
3877 print "\nCreated C -> T converted versions of the FastA file $filename ($count sequences in total)\n\n";
|
|
|
3878 }
|
|
|
3879 else{
|
|
|
3880 print "\nCreated C -> T as well as G -> A converted versions of the FastA file $filename ($count sequences in total)\n\n";
|
|
|
3881 }
|
|
|
3882 return ($C_to_T_infile,$G_to_A_infile);
|
|
|
3883 }
|
|
|
3884
|
|
|
3885 sub biTransformFastAFiles_paired_end {
|
|
|
3886 my ($file,$read_number) = @_;
|
|
|
3887
|
|
|
3888 my ($dir,$filename);
|
|
|
3889 if ($file =~ /\//){
|
|
|
3890 ($dir,$filename) = $file =~ m/(.*\/)(.*)$/;
|
|
|
3891 }
|
|
|
3892 else{
|
|
|
3893 $filename = $file;
|
|
|
3894 }
|
|
|
3895
|
|
|
3896 ### gzipped version of the infile
|
|
|
3897 if ($file =~ /\.gz$/){
|
|
|
3898 open (IN,"zcat $file |") or die "Couldn't read from file $file: $!\n";
|
|
|
3899 }
|
|
|
3900 else{
|
|
|
3901 open (IN,$file) or die "Couldn't read from file $file: $!\n";
|
|
|
3902 }
|
|
|
3903
|
|
|
3904 if ($skip){
|
|
|
3905 warn "Skipping the first $skip reads from $file\n";
|
|
|
3906 sleep (1);
|
|
|
3907 }
|
|
|
3908 if ($upto){
|
|
|
3909 warn "Processing reads up to sequence no. $upto from $file\n";
|
|
|
3910 sleep (1);
|
|
|
3911 }
|
|
|
3912
|
|
|
3913 my $C_to_T_infile = my $G_to_A_infile = $filename;
|
|
|
3914 $C_to_T_infile =~ s/$/_C_to_T.fa/;
|
|
|
3915 $G_to_A_infile =~ s/$/_G_to_A.fa/;
|
|
|
3916
|
|
|
3917 if ($directional){
|
|
|
3918 if ($read_number == 1){
|
|
|
3919 print "Writing a C -> T converted version of the input file $filename to $temp_dir$C_to_T_infile\n";
|
|
|
3920 open (CTOT,'>',"$temp_dir$C_to_T_infile") or die "Couldn't write to file $!\n";
|
|
|
3921 }
|
|
|
3922 elsif ($read_number == 2){
|
|
|
3923 print "Writing a G -> A converted version of the input file $filename to $temp_dir$G_to_A_infile\n";
|
|
|
3924 open (GTOA,'>',"$temp_dir$G_to_A_infile") or die "Couldn't write to file $!\n";
|
|
|
3925 }
|
|
|
3926 else{
|
|
|
3927 die "Read number needs to be 1 or 2, but was: $read_number\n\n";
|
|
|
3928 }
|
|
|
3929 }
|
|
|
3930 else{ # all four strand output
|
|
|
3931 print "Writing a C -> T converted version of the input file $filename to $temp_dir$C_to_T_infile\n";
|
|
|
3932 print "Writing a G -> A converted version of the input file $filename to $temp_dir$G_to_A_infile\n";
|
|
|
3933 open (CTOT,'>',"$temp_dir$C_to_T_infile") or die "Couldn't write to file $!\n";
|
|
|
3934 open (GTOA,'>',"$temp_dir$G_to_A_infile") or die "Couldn't write to file $!\n";
|
|
|
3935 }
|
|
|
3936
|
|
|
3937 my $count = 0;
|
|
|
3938
|
|
|
3939 while (1){
|
|
|
3940 my $header = <IN>;
|
|
|
3941 my $sequence= <IN>;
|
|
|
3942 last unless ($header and $sequence);
|
|
|
3943
|
|
|
3944 $header = fix_IDs($header); # this is to avoid problems with truncated read ID when they contain white spaces
|
|
|
3945
|
|
|
3946 ++$count;
|
|
|
3947
|
|
|
3948 if ($skip){
|
|
|
3949 next unless ($count > $skip);
|
|
|
3950 }
|
|
|
3951 if ($upto){
|
|
|
3952 last if ($count > $upto);
|
|
|
3953 }
|
|
|
3954
|
|
|
3955 $sequence = uc$sequence; # make input file case insensitive
|
|
|
3956
|
|
|
3957 # detecting if the input file contains tab stops, as this is likely to result in no alignments
|
|
|
3958 if (index($header,"\t") != -1){
|
|
|
3959 $seqID_contains_tabs++;
|
|
|
3960 }
|
|
|
3961
|
|
|
3962 ## small check if the sequence seems to be in FastA format
|
|
|
3963 die "Input file doesn't seem to be in FastA format at sequence $count: $!\n" unless ($header =~ /^>.*/);
|
|
|
3964
|
|
|
3965 if ($read_number == 1){
|
|
|
3966 if ($bowtie2){
|
|
|
3967 $header =~ s/$/\/1\/1/;
|
|
|
3968 }
|
|
|
3969 else{
|
|
|
3970 $header =~ s/$/\/1/;
|
|
|
3971 }
|
|
|
3972 }
|
|
|
3973 elsif ($read_number == 2){
|
|
|
3974 if ($bowtie2){
|
|
|
3975 $header =~ s/$/\/2\/2/;
|
|
|
3976 }
|
|
|
3977 else{
|
|
|
3978 $header =~ s/$/\/2/;
|
|
|
3979 }
|
|
|
3980 }
|
|
|
3981 else{
|
|
|
3982 die "Read number needs to be 1 or 2, but was: $read_number\n\n";
|
|
|
3983 }
|
|
|
3984 my $sequence_C_to_T = my $sequence_G_to_A = $sequence;
|
|
|
3985
|
|
|
3986 $sequence_C_to_T =~ tr/C/T/;
|
|
|
3987 $sequence_G_to_A =~ tr/G/A/;
|
|
|
3988
|
|
|
3989 if ($directional){
|
|
|
3990
|
|
|
3991 if ($read_number == 1){
|
|
|
3992 print CTOT "$header$sequence_C_to_T";
|
|
|
3993 }
|
|
|
3994 elsif ($read_number == 2){
|
|
|
3995 print GTOA "$header$sequence_G_to_A";
|
|
|
3996 }
|
|
|
3997 }
|
|
|
3998 else{
|
|
|
3999 print CTOT "$header$sequence_C_to_T";
|
|
|
4000 print GTOA "$header$sequence_G_to_A";
|
|
|
4001 }
|
|
|
4002 }
|
|
|
4003
|
|
|
4004 if ($directional){
|
|
|
4005 if ($read_number == 1){
|
|
|
4006 print "\nCreated C -> T converted version of the FastA file $filename ($count sequences in total)\n\n";
|
|
|
4007 }
|
|
|
4008 else{
|
|
|
4009 print "\nCreated G -> A converted version of the FastA file $filename ($count sequences in total)\n\n";
|
|
|
4010 }
|
|
|
4011 }
|
|
|
4012 else{
|
|
|
4013 print "\nCreated C -> T as well as G -> A converted versions of the FastA file $filename ($count sequences in total)\n\n";
|
|
|
4014 }
|
|
|
4015
|
|
|
4016 if ($directional){
|
|
|
4017 if ($read_number == 1){
|
|
|
4018 return ($C_to_T_infile);
|
|
|
4019 }
|
|
|
4020 else{
|
|
|
4021 return ($G_to_A_infile);
|
|
|
4022 }
|
|
|
4023 }
|
|
|
4024 else{
|
|
|
4025 return ($C_to_T_infile,$G_to_A_infile);
|
|
|
4026 }
|
|
|
4027 }
|
|
|
4028
|
|
|
4029
|
|
|
4030 sub biTransformFastQFiles {
|
|
|
4031 my $file = shift;
|
|
|
4032 my ($dir,$filename);
|
|
|
4033 if ($file =~ /\//){
|
|
|
4034 ($dir,$filename) = $file =~ m/(.*\/)(.*)$/;
|
|
|
4035 }
|
|
|
4036 else{
|
|
|
4037 $filename = $file;
|
|
|
4038 }
|
|
|
4039
|
|
|
4040 ### gzipped version of the infile
|
|
|
4041 if ($file =~ /\.gz$/){
|
|
|
4042 open (IN,"zcat $file |") or die "Couldn't read from file $file: $!\n";
|
|
|
4043 }
|
|
|
4044 else{
|
|
|
4045 open (IN,$file) or die "Couldn't read from file $file: $!\n";
|
|
|
4046 }
|
|
|
4047
|
|
|
4048 if ($skip){
|
|
|
4049 warn "Skipping the first $skip reads from $file\n";
|
|
|
4050 sleep (1);
|
|
|
4051 }
|
|
|
4052 if ($upto){
|
|
|
4053 warn "Processing reads up to sequence no. $upto from $file\n";
|
|
|
4054 sleep (1);
|
|
|
4055 }
|
|
|
4056
|
|
|
4057 my $C_to_T_infile = my $G_to_A_infile = $filename;
|
|
|
4058
|
|
|
4059 $C_to_T_infile =~ s/$/_C_to_T.fastq/;
|
|
|
4060 print "Writing a C -> T converted version of the input file $filename to $temp_dir$C_to_T_infile\n";
|
|
|
4061 open (CTOT,'>',"$temp_dir$C_to_T_infile") or die "Couldn't write to file $!\n";
|
|
|
4062
|
|
|
4063 unless ($directional){
|
|
|
4064 $G_to_A_infile =~ s/$/_G_to_A.fastq/;
|
|
|
4065 print "Writing a G -> A converted version of the input file $filename to $temp_dir$G_to_A_infile\n";
|
|
|
4066 open (GTOA,'>',"$temp_dir$G_to_A_infile") or die "Couldn't write to file $!\n";
|
|
|
4067 }
|
|
|
4068
|
|
|
4069 my $count = 0;
|
|
|
4070 while (1){
|
|
|
4071 my $identifier = <IN>;
|
|
|
4072 my $sequence = <IN>;
|
|
|
4073 my $identifier2 = <IN>;
|
|
|
4074 my $quality_score = <IN>;
|
|
|
4075 last unless ($identifier and $sequence and $identifier2 and $quality_score);
|
|
|
4076
|
|
|
4077 $identifier = fix_IDs($identifier); # this is to avoid problems with truncated read ID when they contain white spaces
|
|
|
4078
|
|
|
4079 ++$count;
|
|
|
4080
|
|
|
4081 if ($skip){
|
|
|
4082 next unless ($count > $skip);
|
|
|
4083 }
|
|
|
4084 if ($upto){
|
|
|
4085 last if ($count > $upto);
|
|
|
4086 }
|
|
|
4087
|
|
|
4088 $sequence = uc$sequence; # make input file case insensitive
|
|
|
4089
|
|
|
4090 # detecting if the input file contains tab stops, as this is likely to result in no alignments
|
|
|
4091 if (index($identifier,"\t") != -1){
|
|
|
4092 $seqID_contains_tabs++;
|
|
|
4093 }
|
|
|
4094
|
|
|
4095 ## small check if the sequence file appears to be a FastQ file
|
|
|
4096 if ($identifier !~ /^\@/ or $identifier2 !~ /^\+/){
|
|
|
4097 die "Input file doesn't seem to be in FastQ format at sequence $count: $!\n";
|
|
|
4098 }
|
|
|
4099
|
|
|
4100 my $sequence_C_to_T = $sequence;
|
|
|
4101 $sequence_C_to_T =~ tr/C/T/;
|
|
|
4102 print CTOT join ('',$identifier,$sequence_C_to_T,$identifier2,$quality_score);
|
|
|
4103
|
|
|
4104 unless ($directional){
|
|
|
4105 my $sequence_G_to_A = $sequence;
|
|
|
4106 $sequence_G_to_A =~ tr/G/A/;
|
|
|
4107 print GTOA join ('',$identifier,$sequence_G_to_A,$identifier2,$quality_score);
|
|
|
4108 }
|
|
|
4109 }
|
|
|
4110
|
|
|
4111 if ($directional){
|
|
|
4112 print "\nCreated C -> T converted versions of the FastQ file $filename ($count sequences in total)\n\n";
|
|
|
4113 }
|
|
|
4114 else{
|
|
|
4115 print "\nCreated C -> T as well as G -> A converted versions of the FastQ file $filename ($count sequences in total)\n\n";
|
|
|
4116 }
|
|
|
4117
|
|
|
4118 return ($C_to_T_infile,$G_to_A_infile);
|
|
|
4119 }
|
|
|
4120
|
|
|
4121 sub biTransformFastQFiles_paired_end {
|
|
|
4122 my ($file,$read_number) = @_;
|
|
|
4123 my ($dir,$filename);
|
|
|
4124
|
|
|
4125 if ($file =~ /\//){
|
|
|
4126 ($dir,$filename) = $file =~ m/(.*\/)(.*)$/;
|
|
|
4127 }
|
|
|
4128 else{
|
|
|
4129 $filename = $file;
|
|
|
4130 }
|
|
|
4131
|
|
|
4132 ### gzipped version of the infile
|
|
|
4133 if ($file =~ /\.gz$/){
|
|
|
4134 open (IN,"zcat $file |") or die "Couldn't read from file $file: $!\n";
|
|
|
4135 }
|
|
|
4136 else{
|
|
|
4137 open (IN,$file) or die "Couldn't read from file $file: $!\n";
|
|
|
4138 }
|
|
|
4139
|
|
|
4140 if ($skip){
|
|
|
4141 warn "Skipping the first $skip reads from $file\n";
|
|
|
4142 sleep (1);
|
|
|
4143 }
|
|
|
4144 if ($upto){
|
|
|
4145 warn "Processing reads up to sequence no. $upto from $file\n";
|
|
|
4146 sleep (1);
|
|
|
4147 }
|
|
|
4148
|
|
|
4149 my $C_to_T_infile = my $G_to_A_infile = $filename;
|
|
|
4150 $C_to_T_infile =~ s/$/_C_to_T.fastq/;
|
|
|
4151 $G_to_A_infile =~ s/$/_G_to_A.fastq/;
|
|
|
4152
|
|
|
4153 if ($directional){
|
|
|
4154 if ($read_number == 1){
|
|
|
4155 print "Writing a C -> T converted version of the input file $filename to $temp_dir$C_to_T_infile\n";
|
|
|
4156 open (CTOT,'>',"$temp_dir$C_to_T_infile") or die "Couldn't write to file $!\n";
|
|
|
4157 }
|
|
|
4158 elsif ($read_number == 2){
|
|
|
4159 print "Writing a G -> A converted version of the input file $filename to $temp_dir$G_to_A_infile\n";
|
|
|
4160 open (GTOA,'>',"$temp_dir$G_to_A_infile") or die "Couldn't write to file $!\n";
|
|
|
4161 }
|
|
|
4162 else{
|
|
|
4163 die "Read number needs to be 1 or 2, but was $read_number!\n\n";
|
|
|
4164 }
|
|
|
4165 }
|
|
|
4166 else{
|
|
|
4167 print "Writing a C -> T converted version of the input file $filename to $temp_dir$C_to_T_infile\n";
|
|
|
4168 print "Writing a G -> A converted version of the input file $filename to $temp_dir$G_to_A_infile\n";
|
|
|
4169 open (CTOT,'>',"$temp_dir$C_to_T_infile") or die "Couldn't write to file $!\n";
|
|
|
4170 open (GTOA,'>',"$temp_dir$G_to_A_infile") or die "Couldn't write to file $!\n";
|
|
|
4171 }
|
|
|
4172
|
|
|
4173 my $count = 0;
|
|
|
4174
|
|
|
4175 while (1){
|
|
|
4176 my $identifier = <IN>;
|
|
|
4177 my $sequence = <IN>;
|
|
|
4178 my $identifier2 = <IN>;
|
|
|
4179 my $quality_score = <IN>;
|
|
|
4180 last unless ($identifier and $sequence and $identifier2 and $quality_score);
|
|
|
4181 ++$count;
|
|
|
4182
|
|
|
4183 $identifier = fix_IDs($identifier); # this is to avoid problems with truncated read ID when they contain white spaces
|
|
|
4184
|
|
|
4185 if ($skip){
|
|
|
4186 next unless ($count > $skip);
|
|
|
4187 }
|
|
|
4188 if ($upto){
|
|
|
4189 last if ($count > $upto);
|
|
|
4190 }
|
|
|
4191
|
|
|
4192 $sequence= uc$sequence; # make input file case insensitive
|
|
|
4193
|
|
|
4194 ## small check if the sequence file appears to be a FastQ file
|
|
|
4195 if ($identifier !~ /^\@/ or $identifier2 !~ /^\+/){
|
|
|
4196 die "Input file doesn't seem to be in FastQ format at sequence $count: $!\n";
|
|
|
4197 }
|
|
|
4198 my $sequence_C_to_T = my $sequence_G_to_A = $sequence;
|
|
|
4199
|
|
|
4200 if ($read_number == 1){
|
|
|
4201 if ($bowtie2){
|
|
|
4202 $identifier =~ s/$/\/1\/1/;
|
|
|
4203 }
|
|
|
4204 else{
|
|
|
4205 $identifier =~ s/$/\/1/;
|
|
|
4206 }
|
|
|
4207 }
|
|
|
4208 elsif ($read_number == 2){
|
|
|
4209 if ($bowtie2){
|
|
|
4210 $identifier =~ s/$/\/2\/2/;
|
|
|
4211 }
|
|
|
4212 else{
|
|
|
4213 $identifier =~ s/$/\/2/;
|
|
|
4214 }
|
|
|
4215 }
|
|
|
4216 else{
|
|
|
4217 die "Read number needs to be 1 or 2\n";
|
|
|
4218 }
|
|
|
4219
|
|
|
4220 $sequence_C_to_T =~ tr/C/T/;
|
|
|
4221 $sequence_G_to_A =~ tr/G/A/;
|
|
|
4222
|
|
|
4223 if ($directional){
|
|
|
4224 if ($read_number == 1){
|
|
|
4225 print CTOT join ('',$identifier,$sequence_C_to_T,$identifier2,$quality_score);
|
|
|
4226 }
|
|
|
4227 else{
|
|
|
4228 print GTOA join ('',$identifier,$sequence_G_to_A,$identifier2,$quality_score);
|
|
|
4229 }
|
|
|
4230 }
|
|
|
4231 else{
|
|
|
4232 print CTOT join ('',$identifier,$sequence_C_to_T,$identifier2,$quality_score);
|
|
|
4233 print GTOA join ('',$identifier,$sequence_G_to_A,$identifier2,$quality_score);
|
|
|
4234 }
|
|
|
4235 }
|
|
|
4236
|
|
|
4237 if ($directional){
|
|
|
4238 if ($read_number == 1){
|
|
|
4239 print "\nCreated C -> T converted version of the FastQ file $filename ($count sequences in total)\n\n";
|
|
|
4240 }
|
|
|
4241 else{
|
|
|
4242 print "\nCreated G -> A converted version of the FastQ file $filename ($count sequences in total)\n\n";
|
|
|
4243 }
|
|
|
4244 }
|
|
|
4245 else{
|
|
|
4246 print "\nCreated C -> T as well as G -> A converted versions of the FastQ file $filename ($count sequences in total)\n\n";
|
|
|
4247 }
|
|
|
4248 if ($directional){
|
|
|
4249 if ($read_number == 1){
|
|
|
4250 return ($C_to_T_infile);
|
|
|
4251 }
|
|
|
4252 else{
|
|
|
4253 return ($G_to_A_infile);
|
|
|
4254 }
|
|
|
4255 }
|
|
|
4256 else{
|
|
|
4257 return ($C_to_T_infile,$G_to_A_infile);
|
|
|
4258 }
|
|
|
4259 }
|
|
|
4260
|
|
|
4261 sub fix_IDs{
|
|
|
4262 my $id = shift;
|
|
|
4263 $id =~ s/[ \t]+/_/g; # replace spaces or tabs with underscores
|
|
|
4264 return $id;
|
|
|
4265 }
|
|
|
4266
|
|
|
4267 sub ensure_sensical_alignment_orientation_single_end{
|
|
|
4268 my $index = shift; # index number if the sequence produced an alignment
|
|
|
4269 my $strand = shift;
|
|
|
4270 ### setting $orientation to 1 if it is in the correct orientation, and leave it 0 if it is the nonsensical wrong one
|
|
|
4271 my $orientation = 0;
|
|
|
4272 ##############################################################################################################
|
|
|
4273 ## FORWARD converted read against FORWARD converted genome (read: C->T.....C->T.. genome:C->T.......C->T)
|
|
|
4274 ## here we only want reads in the forward (+) orientation
|
|
|
4275 if ($fhs[$index]->{name} eq 'CTreadCTgenome') {
|
|
|
4276 ### if the alignment is (+) we count it, and return 1 for a correct orientation
|
|
|
4277 if ($strand eq '+') {
|
|
|
4278 $fhs[$index]->{seen}++;
|
|
|
4279 $orientation = 1;
|
|
|
4280 return $orientation;
|
|
|
4281 }
|
|
|
4282 ### if the orientation equals (-) the alignment is nonsensical
|
|
|
4283 elsif ($strand eq '-') {
|
|
|
4284 $fhs[$index]->{wrong_strand}++;
|
|
|
4285 return $orientation;
|
|
|
4286 }
|
|
|
4287 }
|
|
|
4288 ###############################################################################################################
|
|
|
4289 ## FORWARD converted read against reverse converted genome (read: C->T.....C->T.. genome: G->A.......G->A)
|
|
|
4290 ## here we only want reads in the forward (-) orientation
|
|
|
4291 elsif ($fhs[$index]->{name} eq 'CTreadGAgenome') {
|
|
|
4292 ### if the alignment is (-) we count it and return 1 for a correct orientation
|
|
|
4293 if ($strand eq '-') {
|
|
|
4294 $fhs[$index]->{seen}++;
|
|
|
4295 $orientation = 1;
|
|
|
4296 return $orientation;
|
|
|
4297 }
|
|
|
4298 ### if the orientation equals (+) the alignment is nonsensical
|
|
|
4299 elsif ($strand eq '+') {
|
|
|
4300 $fhs[$index]->{wrong_strand}++;
|
|
|
4301 return $orientation;
|
|
|
4302 }
|
|
|
4303 }
|
|
|
4304 ###############################################################################################################
|
|
|
4305 ## Reverse converted read against FORWARD converted genome (read: G->A.....G->A.. genome: C->T.......C->T)
|
|
|
4306 ## here we only want reads in the forward (-) orientation
|
|
|
4307 elsif ($fhs[$index]->{name} eq 'GAreadCTgenome') {
|
|
|
4308 ### if the alignment is (-) we count it and return 1 for a correct orientation
|
|
|
4309 if ($strand eq '-') {
|
|
|
4310 $fhs[$index]->{seen}++;
|
|
|
4311 $orientation = 1;
|
|
|
4312 return $orientation;
|
|
|
4313 }
|
|
|
4314 ### if the orientation equals (+) the alignment is nonsensical
|
|
|
4315 elsif ($strand eq '+') {
|
|
|
4316 $fhs[$index]->{wrong_strand}++;
|
|
|
4317 return $orientation;
|
|
|
4318 }
|
|
|
4319 }
|
|
|
4320 ###############################################################################################################
|
|
|
4321 ## Reverse converted read against reverse converted genome (read: G->A.....G->A.. genome: G->A.......G->A)
|
|
|
4322 ## here we only want reads in the forward (+) orientation
|
|
|
4323 elsif ($fhs[$index]->{name} eq 'GAreadGAgenome') {
|
|
|
4324 ### if the alignment is (+) we count it and return 1 for a correct orientation
|
|
|
4325 if ($strand eq '+') {
|
|
|
4326 $fhs[$index]->{seen}++;
|
|
|
4327 $orientation = 1;
|
|
|
4328 return $orientation;
|
|
|
4329 }
|
|
|
4330 ### if the orientation equals (-) the alignment is nonsensical
|
|
|
4331 elsif ($strand eq '-') {
|
|
|
4332 $fhs[$index]->{wrong_strand}++;
|
|
|
4333 return $orientation;
|
|
|
4334 }
|
|
|
4335 } else{
|
|
|
4336 die "One of the above conditions must be true\n";
|
|
|
4337 }
|
|
|
4338 }
|
|
|
4339
|
|
|
4340 sub ensure_sensical_alignment_orientation_paired_ends{
|
|
|
4341 my ($index,$id_1,$strand_1,$id_2,$strand_2) = @_; # index number if the sequence produced an alignment
|
|
|
4342 ### setting $orientation to 1 if it is in the correct orientation, and leave it 0 if it is the nonsensical wrong one
|
|
|
4343 my $orientation = 0;
|
|
|
4344 ##############################################################################################################
|
|
|
4345 ## [Index 0, sequence originated from (converted) forward strand]
|
|
|
4346 ## CT converted read 1
|
|
|
4347 ## GA converted read 2
|
|
|
4348 ## CT converted genome
|
|
|
4349 ## here we only want read 1 in (+) orientation and read 2 in (-) orientation
|
|
|
4350 if ($fhs[$index]->{name} eq 'CTread1GAread2CTgenome') {
|
|
|
4351 ### if the paired-end alignment is read1 (+) and read2 (-) we count it, and return 1 for a correct orientation
|
|
|
4352 if ($id_1 =~ /1$/ and $strand_1 eq '+' and $id_2 =~ /2$/ and $strand_2 eq '-') {
|
|
|
4353 $fhs[$index]->{seen}++;
|
|
|
4354 $orientation = 1;
|
|
|
4355 return $orientation;
|
|
|
4356 }
|
|
|
4357 ### if the read 2 is in (+) orientation and read 1 in (-) the alignment is nonsensical
|
|
|
4358 elsif ($id_1 =~ /2$/ and $strand_1 eq '+' and $id_2 =~ /1$/ and $strand_2 eq '-') {
|
|
|
4359 $fhs[$index]->{wrong_strand}++;
|
|
|
4360 return $orientation;
|
|
|
4361 }
|
|
|
4362 else{
|
|
|
4363 die "id1: $id_1\tid2: $id_2\tThis should be impossible\n";
|
|
|
4364 }
|
|
|
4365 }
|
|
|
4366 ###############################################################################################################
|
|
|
4367 ## [Index 1, sequence originated from (converted) reverse strand]
|
|
|
4368 ## GA converted read 1
|
|
|
4369 ## CT converted read 2
|
|
|
4370 ## GA converted genome
|
|
|
4371 ## here we only want read 1 in (+) orientation and read 2 in (-) orientation
|
|
|
4372 elsif ($fhs[$index]->{name} eq 'GAread1CTread2GAgenome') {
|
|
|
4373 ### if the paired-end alignment is read1 (+) and read2 (-) we count it, and return 1 for a correct orientation
|
|
|
4374 if ($id_1 =~ /1$/ and $strand_1 eq '+' and $id_2 =~ /2$/ and $strand_2 eq '-') {
|
|
|
4375 $fhs[$index]->{seen}++;
|
|
|
4376 $orientation = 1;
|
|
|
4377 return $orientation;
|
|
|
4378 }
|
|
|
4379 ### if the read 2 is in (+) orientation and read 1 in (-) the alignment is nonsensical
|
|
|
4380 elsif ($id_1 =~ /2$/ and $strand_1 eq '+' and $id_2 =~ /1$/ and $strand_2 eq '-') {
|
|
|
4381 $fhs[$index]->{wrong_strand}++;
|
|
|
4382 return $orientation;
|
|
|
4383 }
|
|
|
4384 else{
|
|
|
4385 die "id1: $id_1\tid2: $id_2\tThis should be impossible\n";
|
|
|
4386 }
|
|
|
4387 }
|
|
|
4388 ###############################################################################################################
|
|
|
4389 ## [Index 2, sequence originated from complementary to (converted) forward strand]
|
|
|
4390 ## GA converted read 1
|
|
|
4391 ## CT converted read 2
|
|
|
4392 ## CT converted genome
|
|
|
4393 ## here we only want read 1 in (-) orientation and read 2 in (+) orientation
|
|
|
4394 elsif ($fhs[$index]->{name} eq 'GAread1CTread2CTgenome') {
|
|
|
4395 ### if the paired-end alignment is read1 (-) and read2 (+) we count it, and return 1 for a correct orientation
|
|
|
4396 if ($id_1 =~ /2$/ and $strand_1 eq '+' and $id_2 =~ /1$/ and $strand_2 eq '-') {
|
|
|
4397 $fhs[$index]->{seen}++;
|
|
|
4398 $orientation = 1;
|
|
|
4399 return $orientation;
|
|
|
4400 }
|
|
|
4401 ### if the read 2 is in (+) orientation and read 1 in (-) the alignment is nonsensical
|
|
|
4402 elsif ($id_1 =~ /1$/ and $strand_1 eq '+' and $id_2 =~ /2$/ and $strand_2 eq '-') {
|
|
|
4403 $fhs[$index]->{wrong_strand}++;
|
|
|
4404 return $orientation;
|
|
|
4405 }
|
|
|
4406 else{
|
|
|
4407 die "id1: $id_1\tid2: $id_2\tThis should be impossible\n";
|
|
|
4408 }
|
|
|
4409 }
|
|
|
4410 ###############################################################################################################
|
|
|
4411 ## [Index 3, sequence originated from complementary to (converted) reverse strand]
|
|
|
4412 ## CT converted read 1
|
|
|
4413 ## GA converted read 2
|
|
|
4414 ## GA converted genome
|
|
|
4415 ## here we only want read 1 in (+) orientation and read 2 in (-) orientation
|
|
|
4416 elsif ($fhs[$index]->{name} eq 'CTread1GAread2GAgenome') {
|
|
|
4417 ### if the paired-end alignment is read1 (-) and read2 (+) we count it, and return 1 for a correct orientation
|
|
|
4418 if ($id_1 =~ /2$/ and $strand_1 eq '+' and $id_2 =~ /1$/ and $strand_2 eq '-') {
|
|
|
4419 $fhs[$index]->{seen}++;
|
|
|
4420 $orientation = 1;
|
|
|
4421 return $orientation;
|
|
|
4422 }
|
|
|
4423 ### if the read 2 is in (+) orientation and read 1 in (-) the alignment is nonsensical
|
|
|
4424 elsif ($id_1 =~ /1$/ and $strand_1 eq '+' and $id_2 =~ /2$/ and $strand_2 eq '-') {
|
|
|
4425 $fhs[$index]->{wrong_strand}++;
|
|
|
4426 return $orientation;
|
|
|
4427 }
|
|
|
4428 else{
|
|
|
4429 die "id1: $id_1\tid2: $id_2\tThis should be impossible\n";
|
|
|
4430 }
|
|
|
4431 }
|
|
|
4432 else{
|
|
|
4433 die "One of the above conditions must be true\n";
|
|
|
4434 }
|
|
|
4435 }
|
|
|
4436
|
|
|
4437 #####################################################################################################################################################
|
|
|
4438
|
|
|
4439 ### Bowtie 1 (default) | PAIRED-END | FASTA
|
|
|
4440
|
|
|
4441 sub paired_end_align_fragments_to_bisulfite_genome_fastA {
|
|
|
4442
|
|
|
4443 my ($C_to_T_infile_1,$G_to_A_infile_1,$C_to_T_infile_2,$G_to_A_infile_2) = @_;
|
|
|
4444
|
|
|
4445 if ($directional){
|
|
|
4446 print "Input files are $C_to_T_infile_1 and $G_to_A_infile_2 (FastA)\n";
|
|
|
4447 }
|
|
|
4448 else{
|
|
|
4449 print "Input files are $C_to_T_infile_1 and $G_to_A_infile_1 and $C_to_T_infile_2 and $G_to_A_infile_2 (FastA)\n";
|
|
|
4450 }
|
|
|
4451
|
|
|
4452 ## Now starting up to 4 instances of Bowtie feeding in the converted sequence files and reading in the first line of the bowtie output, and storing it in the
|
|
|
4453 ## data structure above
|
|
|
4454 if ($directional){
|
|
|
4455 warn "Now running 2 instances of Bowtie against the bisulfite genome of $genome_folder with the specified options: $bowtie_options\n\n";
|
|
|
4456 }
|
|
|
4457 else{
|
|
|
4458 warn "Now running 4 individual instances of Bowtie against the bisulfite genome of $genome_folder with the specified options: $bowtie_options\n\n";
|
|
|
4459 }
|
|
|
4460
|
|
|
4461 foreach my $fh (@fhs) {
|
|
|
4462
|
|
|
4463 if ($directional){
|
|
|
4464 unless ($fh->{inputfile_1}){
|
|
|
4465 $fh->{last_seq_id} = undef;
|
|
|
4466 $fh->{last_line_1} = undef;
|
|
|
4467 $fh->{last_line_2} = undef;
|
|
|
4468 next;
|
|
|
4469 }
|
|
|
4470 }
|
|
|
4471
|
|
|
4472 my $bt_options = $bowtie_options;
|
|
|
4473 if ($fh->{name} eq 'CTread1GAread2CTgenome' or $fh->{name} eq 'GAread1CTread2GAgenome'){
|
|
|
4474 $bt_options .= ' --norc'; ### ensuring the alignments are only reported in a sensible manner
|
|
|
4475 }
|
|
|
4476 else {
|
|
|
4477 $bt_options .= ' --nofw';
|
|
|
4478 }
|
|
|
4479
|
|
|
4480 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: $bt_options)\n";
|
|
|
4481 open ($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: $!";
|
|
|
4482
|
|
|
4483 my $line_1 = $fh->{fh}->getline();
|
|
|
4484 my $line_2 = $fh->{fh}->getline();
|
|
|
4485
|
|
|
4486 # if Bowtie produces an alignment we store the first line of the output
|
|
|
4487 if ($line_1 and $line_2) {
|
|
|
4488 chomp $line_1;
|
|
|
4489 chomp $line_2;
|
|
|
4490 my $id_1 = (split(/\t/,$line_1))[0]; # this is the first element of the first bowtie output line (= the sequence identifier)
|
|
|
4491 my $id_2 = (split(/\t/,$line_2))[0]; # this is the first element of the second bowtie output line
|
|
|
4492
|
|
|
4493 ### Bowtie always reports the alignment with the smaller chromosomal position first. This can be either sequence 1 or sequence 2.
|
|
|
4494 ### We will thus identify which sequence was read 1 and store this ID as last_seq_id
|
|
|
4495
|
|
|
4496 if ($id_1 =~ s/\/1$//){ # removing the read 1 tag if present
|
|
|
4497 $fh->{last_seq_id} = $id_1;
|
|
|
4498 }
|
|
|
4499 elsif ($id_2 =~ s/\/1$//){ # removing the read 1 tag if present
|
|
|
4500 $fh->{last_seq_id} = $id_2;
|
|
|
4501 }
|
|
|
4502 else{
|
|
|
4503 die "Either the first or the second id need to be read 1! ID1 was: $id_1; ID2 was: $id_2\n";
|
|
|
4504 }
|
|
|
4505
|
|
|
4506 $fh->{last_line_1} = $line_1; # this contains either read 1 or read 2
|
|
|
4507 $fh->{last_line_2} = $line_2; # this contains either read 1 or read 2
|
|
|
4508 warn "Found first alignment:\n$fh->{last_line_1}\n$fh->{last_line_2}\n";
|
|
|
4509 }
|
|
|
4510 # otherwise we just initialise last_seq_id and last_lines as undefined
|
|
|
4511 else {
|
|
|
4512 print "Found no alignment, assigning undef to last_seq_id and last_lines\n";
|
|
|
4513 $fh->{last_seq_id} = undef;
|
|
|
4514 $fh->{last_line_1} = undef;
|
|
|
4515 $fh->{last_line_2} = undef;
|
|
|
4516 }
|
|
|
4517 }
|
|
|
4518 }
|
|
|
4519
|
|
|
4520 ### Bowtie 2 | PAIRED-END | FASTA
|
|
|
4521
|
|
|
4522 sub paired_end_align_fragments_to_bisulfite_genome_fastA_bowtie2 {
|
|
|
4523 my ($C_to_T_infile_1,$G_to_A_infile_1,$C_to_T_infile_2,$G_to_A_infile_2) = @_;
|
|
|
4524 if ($directional){
|
|
|
4525 print "Input files are $C_to_T_infile_1 and $G_to_A_infile_2 (FastA)\n";
|
|
|
4526 }
|
|
|
4527 else{
|
|
|
4528 print "Input files are $C_to_T_infile_1 and $G_to_A_infile_1 and $C_to_T_infile_2 and $G_to_A_infile_2 (FastA)\n";
|
|
|
4529 }
|
|
|
4530
|
|
|
4531 ## Now starting up to 4 instances of Bowtie feeding in the converted sequence files and reading in the first line of the bowtie output, and storing it in the
|
|
|
4532 ## data structure above
|
|
|
4533 if ($directional){
|
|
|
4534 warn "Now running 2 instances of Bowtie 2 against the bisulfite genome of $genome_folder with the specified options: $bowtie_options\n\n";
|
|
|
4535 }
|
|
|
4536 else{
|
|
|
4537 warn "Now running 4 individual instances of Bowtie 2 against the bisulfite genome of $genome_folder with the specified options: $bowtie_options\n\n";
|
|
|
4538 }
|
|
|
4539
|
|
|
4540 foreach my $fh (@fhs) {
|
|
|
4541
|
|
|
4542 if ($directional){
|
|
|
4543 unless ($fh->{inputfile_1}){
|
|
|
4544 $fh->{last_seq_id} = undef;
|
|
|
4545 $fh->{last_line_1} = undef;
|
|
|
4546 $fh->{last_line_2} = undef;
|
|
|
4547 next;
|
|
|
4548 }
|
|
|
4549 }
|
|
|
4550
|
|
|
4551 my $bt2_options = $bowtie_options;
|
|
|
4552 if ($fh->{name} eq 'CTread1GAread2CTgenome' or $fh->{name} eq 'GAread1CTread2GAgenome'){
|
|
|
4553 $bt2_options .= ' --norc'; ### ensuring the alignments are only reported in a sensible manner
|
|
|
4554 }
|
|
|
4555 else {
|
|
|
4556 $bt2_options .= ' --nofw';
|
|
|
4557 }
|
|
|
4558
|
|
|
4559 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: $bt2_options))\n";
|
|
|
4560 open ($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: $!";
|
|
|
4561
|
|
|
4562 ### Bowtie 2 outputs out SAM format, so we need to skip everything until the first sequence
|
|
|
4563 while (1){
|
|
|
4564 $_ = $fh->{fh}->getline();
|
|
|
4565 if ($_) {
|
|
|
4566 last unless ($_ =~ /^\@/); # SAM headers start with @
|
|
|
4567 }
|
|
|
4568 else{
|
|
|
4569 last; # no alignment output
|
|
|
4570 }
|
|
|
4571 }
|
|
|
4572
|
|
|
4573 my $line_1 = $_;
|
|
|
4574 my $line_2 = $fh->{fh}->getline();
|
|
|
4575
|
|
|
4576 # if Bowtie produces an alignment we store the first line of the output
|
|
|
4577 if ($line_1 and $line_2) {
|
|
|
4578 chomp $line_1;
|
|
|
4579 chomp $line_2;
|
|
|
4580 my $id_1 = (split(/\t/,$line_1))[0]; # this is the first element of the first bowtie output line (= the sequence identifier)
|
|
|
4581 my $id_2 = (split(/\t/,$line_2))[0]; # this is the first element of the second bowtie output line
|
|
|
4582
|
|
|
4583 ### Bowtie always reports the alignment with the smaller chromosomal position first. This can be either sequence 1 or sequence 2.
|
|
|
4584 ### We will thus identify which sequence was read 1 and store this ID as last_seq_id
|
|
|
4585
|
|
|
4586 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
|
|
|
4587 $fh->{last_seq_id} = $id_1;
|
|
|
4588 }
|
|
|
4589 elsif ($id_2 =~ s/\/1$//){ # removing the read 1 /2 tag if present
|
|
|
4590 $fh->{last_seq_id} = $id_2;
|
|
|
4591 }
|
|
|
4592 else{
|
|
|
4593 warn "Either the first or the second id need to be read 1! ID1 was: $id_1; ID2 was: $id_2\n";
|
|
|
4594 }
|
|
|
4595
|
|
|
4596 $fh->{last_line_1} = $line_1; # this contains either read 1 or read 2
|
|
|
4597 $fh->{last_line_2} = $line_2; # this contains either read 1 or read 2
|
|
|
4598 warn "Found first alignment:\n$fh->{last_line_1}\n$fh->{last_line_2}\n";
|
|
|
4599 }
|
|
|
4600 # otherwise we just initialise last_seq_id and last_lines as undefined
|
|
|
4601 else {
|
|
|
4602 print "Found no alignment, assigning undef to last_seq_id and last_lines\n";
|
|
|
4603 $fh->{last_seq_id} = undef;
|
|
|
4604 $fh->{last_line_1} = undef;
|
|
|
4605 $fh->{last_line_2} = undef;
|
|
|
4606 }
|
|
|
4607 }
|
|
|
4608 }
|
|
|
4609
|
|
|
4610 ### Bowtie 1 (default) | PAIRED-END | FASTQ
|
|
|
4611
|
|
|
4612 sub paired_end_align_fragments_to_bisulfite_genome_fastQ {
|
|
|
4613 my ($C_to_T_infile_1,$G_to_A_infile_1,$C_to_T_infile_2,$G_to_A_infile_2) = @_;
|
|
|
4614 if ($directional){
|
|
|
4615 print "Input files are $C_to_T_infile_1 $G_to_A_infile_2 (FastQ)\n";
|
|
|
4616 }
|
|
|
4617 else{
|
|
|
4618 print "Input files are $C_to_T_infile_1 and $G_to_A_infile_1 and $C_to_T_infile_2 and $G_to_A_infile_2 (FastQ)\n";
|
|
|
4619 }
|
|
|
4620
|
|
|
4621 ## Now starting up to 4 instances of Bowtie feeding in the converted sequence files and reading in the first line of the bowtie output, and storing it in the
|
|
|
4622 ## data structure above
|
|
|
4623 if ($directional){
|
|
|
4624 warn "Now running 2 instances of Bowtie against the bisulfite genome of $genome_folder with the specified options: $bowtie_options\n\n";
|
|
|
4625 }
|
|
|
4626 else{
|
|
|
4627 warn "Now running 4 individual instances of Bowtie against the bisulfite genome of $genome_folder with the specified options: $bowtie_options\n\n";
|
|
|
4628 }
|
|
|
4629
|
|
|
4630 foreach my $fh (@fhs) {
|
|
|
4631
|
|
|
4632 if ($directional){
|
|
|
4633 unless ($fh->{inputfile_1}){
|
|
|
4634 $fh->{last_seq_id} = undef;
|
|
|
4635 $fh->{last_line_1} = undef;
|
|
|
4636 $fh->{last_line_2} = undef;
|
|
|
4637 next;
|
|
|
4638 }
|
|
|
4639 }
|
|
|
4640
|
|
|
4641 my $bt_options = $bowtie_options;
|
|
|
4642 if ($fh->{name} eq 'CTread1GAread2CTgenome' or $fh->{name} eq 'GAread1CTread2GAgenome'){
|
|
|
4643 $bt_options .= ' --norc'; ### ensuring the alignments are only reported in a sensible manner
|
|
|
4644 }
|
|
|
4645 else {
|
|
|
4646 $bt_options .= ' --nofw';
|
|
|
4647 }
|
|
|
4648
|
|
|
4649 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: $bt_options))\n";
|
|
|
4650 open ($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: $!";
|
|
|
4651
|
|
|
4652 my $line_1 = $fh->{fh}->getline();
|
|
|
4653 my $line_2 = $fh->{fh}->getline();
|
|
|
4654
|
|
|
4655 # if Bowtie produces an alignment we store the first line of the output
|
|
|
4656 if ($line_1 and $line_2) {
|
|
|
4657 chomp $line_1;
|
|
|
4658 chomp $line_2;
|
|
|
4659 ### Bowtie always reports the alignment with the smaller chromosomal position first. This can be either sequence 1 or sequence 2.
|
|
|
4660 ### We will thus identify which sequence was read 1 and store this ID as last_seq_id
|
|
|
4661
|
|
|
4662 my $id_1 = (split(/\t/,$line_1))[0]; # this is the first element of the first bowtie output line (= the sequence identifier)
|
|
|
4663 my $id_2 = (split(/\t/,$line_2))[0]; # this is the first element of the second bowtie output line
|
|
|
4664
|
|
|
4665 if ($id_1 =~ s/\/1$//){ # removing the read 1 tag if present
|
|
|
4666 $fh->{last_seq_id} = $id_1;
|
|
|
4667 }
|
|
|
4668 elsif ($id_2 =~ s/\/1$//){ # removing the read 1 tag if present
|
|
|
4669 $fh->{last_seq_id} = $id_2;
|
|
|
4670 }
|
|
|
4671 else{
|
|
|
4672 die "Either the first or the second id need to be read 1! ID1 was: $id_1; ID2 was: $id_2\n";
|
|
|
4673 }
|
|
|
4674
|
|
|
4675 $fh->{last_line_1} = $line_1; # this contains read 1 or read 2
|
|
|
4676 $fh->{last_line_2} = $line_2; # this contains read 1 or read 2
|
|
|
4677 warn "Found first alignment:\n$fh->{last_line_1}\n$fh->{last_line_2}\n";
|
|
|
4678 }
|
|
|
4679
|
|
|
4680 # otherwise we just initialise last_seq_id and last_lines as undefined
|
|
|
4681 else {
|
|
|
4682 print "Found no alignment, assigning undef to last_seq_id and last_lines\n";
|
|
|
4683 $fh->{last_seq_id} = undef;
|
|
|
4684 $fh->{last_line_1} = undef;
|
|
|
4685 $fh->{last_line_2} = undef;
|
|
|
4686 }
|
|
|
4687 }
|
|
|
4688 }
|
|
|
4689
|
|
|
4690 ### Bowtie 2 | PAIRED-END | FASTQ
|
|
|
4691
|
|
|
4692 sub paired_end_align_fragments_to_bisulfite_genome_fastQ_bowtie2 {
|
|
|
4693 my ($C_to_T_infile_1,$G_to_A_infile_1,$C_to_T_infile_2,$G_to_A_infile_2) = @_;
|
|
|
4694 if ($directional){
|
|
|
4695 print "Input files are $C_to_T_infile_1 and $G_to_A_infile_2 (FastQ)\n";
|
|
|
4696 }
|
|
|
4697 else{
|
|
|
4698 print "Input files are $C_to_T_infile_1 and $G_to_A_infile_1 and $C_to_T_infile_2 and $G_to_A_infile_2 (FastQ)\n";
|
|
|
4699 }
|
|
|
4700
|
|
|
4701 ## Now starting up 4 instances of Bowtie 2 feeding in the converted sequence files and reading in the first line of the bowtie output, and storing it in the
|
|
|
4702 ## data structure above
|
|
|
4703 if ($directional){
|
|
|
4704 warn "Now running 2 instances of Bowtie 2 against the bisulfite genome of $genome_folder with the specified options: $bowtie_options\n\n";
|
|
|
4705 }
|
|
|
4706 else{
|
|
|
4707 warn "Now running 4 individual instances of Bowtie 2 against the bisulfite genome of $genome_folder with the specified options: $bowtie_options\n\n";
|
|
|
4708 }
|
|
|
4709
|
|
|
4710 foreach my $fh (@fhs) {
|
|
|
4711
|
|
|
4712 if ($directional){
|
|
|
4713 unless ($fh->{inputfile_1}){
|
|
|
4714 $fh->{last_seq_id} = undef;
|
|
|
4715 $fh->{last_line_1} = undef;
|
|
|
4716 $fh->{last_line_2} = undef;
|
|
|
4717 next;
|
|
|
4718 }
|
|
|
4719 }
|
|
|
4720
|
|
|
4721 my $bt2_options = $bowtie_options;
|
|
|
4722 if ($fh->{name} eq 'CTread1GAread2CTgenome' or $fh->{name} eq 'GAread1CTread2GAgenome'){
|
|
|
4723 $bt2_options .= ' --norc'; ### ensuring the alignments are only reported in a sensible manner
|
|
|
4724 }
|
|
|
4725 else {
|
|
|
4726 $bt2_options .= ' --nofw';
|
|
|
4727 }
|
|
|
4728
|
|
|
4729 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: $bt2_options))\n";
|
|
|
4730 open ($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: $!";
|
|
|
4731
|
|
|
4732 ### Bowtie 2 outputs out SAM format, so we need to skip everything until the first sequence
|
|
|
4733 while (1){
|
|
|
4734 $_ = $fh->{fh}->getline();
|
|
|
4735 if ($_) {
|
|
|
4736 last unless ($_ =~ /^\@/); # SAM headers start with @
|
|
|
4737 }
|
|
|
4738 else{
|
|
|
4739 last; # no alignment output
|
|
|
4740 }
|
|
|
4741 }
|
|
|
4742
|
|
|
4743 my $line_1 = $_;
|
|
|
4744 my $line_2 = $fh->{fh}->getline();
|
|
|
4745
|
|
|
4746 # if Bowtie produces an alignment we store the first line of the output
|
|
|
4747 if ($line_1 and $line_2) {
|
|
|
4748 chomp $line_1;
|
|
|
4749 chomp $line_2;
|
|
|
4750 ### Bowtie always reports the alignment with the smaller chromosomal position first. This can be either sequence 1 or sequence 2.
|
|
|
4751 ### We will thus identify which sequence was read 1 and store this ID as last_seq_id
|
|
|
4752
|
|
|
4753 my $id_1 = (split(/\t/,$line_1))[0]; # this is the first element of the first bowtie output line (= the sequence identifier)
|
|
|
4754 my $id_2 = (split(/\t/,$line_2))[0]; # this is the first element of the second bowtie output line
|
|
|
4755
|
|
|
4756 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
|
|
|
4757 $fh->{last_seq_id} = $id_1;
|
|
|
4758 }
|
|
|
4759 elsif ($id_2 =~ s/\/1$//){ # removing the read 1 tag if present
|
|
|
4760 $fh->{last_seq_id} = $id_2;
|
|
|
4761 }
|
|
|
4762 else{
|
|
|
4763 die "Either the first or the second id need to be read 1! ID1 was: $id_1; ID2 was: $id_2\n";
|
|
|
4764 }
|
|
|
4765
|
|
|
4766 $fh->{last_line_1} = $line_1; # this contains read 1 or read 2
|
|
|
4767 $fh->{last_line_2} = $line_2; # this contains read 1 or read 2
|
|
|
4768 warn "Found first alignment:\n$fh->{last_line_1}\n$fh->{last_line_2}\n";
|
|
|
4769 }
|
|
|
4770
|
|
|
4771 # otherwise we just initialise last_seq_id and last_lines as undefined
|
|
|
4772 else {
|
|
|
4773 print "Found no alignment, assigning undef to last_seq_id and last_lines\n";
|
|
|
4774 $fh->{last_seq_id} = undef;
|
|
|
4775 $fh->{last_line_1} = undef;
|
|
|
4776 $fh->{last_line_2} = undef;
|
|
|
4777 }
|
|
|
4778 }
|
|
|
4779 }
|
|
|
4780
|
|
|
4781 #####################################################################################################################################################
|
|
|
4782
|
|
|
4783 ### Bowtie 1 (default) | SINGLE-END | FASTA
|
|
|
4784 sub single_end_align_fragments_to_bisulfite_genome_fastA {
|
|
|
4785 my ($C_to_T_infile,$G_to_A_infile) = @_;
|
|
|
4786 if ($directional){
|
|
|
4787 print "Input file is $C_to_T_infile (FastA)\n";
|
|
|
4788 }
|
|
|
4789 else{
|
|
|
4790 print "Input files are $C_to_T_infile and $G_to_A_infile (FastA)\n";
|
|
|
4791 }
|
|
|
4792
|
|
|
4793 ## Now starting up to 4 instances of Bowtie feeding in the converted sequence files and reading in the first line of the bowtie output, and storing it in
|
|
|
4794 ## data structure above
|
|
|
4795 if ($directional){
|
|
|
4796 warn "Now running 2 instances of Bowtie against the bisulfite genome of $genome_folder with the specified options: $bowtie_options\n\n";
|
|
|
4797 }
|
|
|
4798 else{
|
|
|
4799 warn "Now running 4 individual instances of Bowtie against the bisulfite genome of $genome_folder with the specified options: $bowtie_options\n\n";
|
|
|
4800 }
|
|
|
4801
|
|
|
4802 foreach my $fh (@fhs) {
|
|
|
4803
|
|
|
4804 my $bt_options = $bowtie_options;
|
|
|
4805 if ($fh->{name} eq 'CTreadCTgenome' or $fh->{name} eq 'GAreadGAgenome'){
|
|
|
4806 $bt_options .= ' --norc'; ### ensuring the alignments are only reported in a sensible manner
|
|
|
4807 }
|
|
|
4808 else {
|
|
|
4809 $bt_options .= ' --nofw';
|
|
|
4810 }
|
|
|
4811
|
|
|
4812 warn "Now starting the Bowtie aligner for $fh->{name} (reading in sequences from $temp_dir$fh->{inputfile} with options: $bt_options)\n";
|
|
|
4813 open ($fh->{fh},"$path_to_bowtie $bt_options $fh->{bisulfiteIndex} $temp_dir$fh->{inputfile} |") or die "Can't open pipe to bowtie: $!";
|
|
|
4814
|
|
|
4815 # if Bowtie produces an alignment we store the first line of the output
|
|
|
4816 $_ = $fh->{fh}->getline();
|
|
|
4817 if ($_) {
|
|
|
4818 chomp;
|
|
|
4819 my $id = (split(/\t/))[0]; # this is the first element of the bowtie output (= the sequence identifier)
|
|
|
4820 $fh->{last_seq_id} = $id;
|
|
|
4821 $fh->{last_line} = $_;
|
|
|
4822 warn "Found first alignment:\t$fh->{last_line}\n";
|
|
|
4823 }
|
|
|
4824 # otherwise we just initialise last_seq_id and last_line as undefined
|
|
|
4825 else {
|
|
|
4826 print "Found no alignment, assigning undef to last_seq_id and last_line\n";
|
|
|
4827 $fh->{last_seq_id} = undef;
|
|
|
4828 $fh->{last_line} = undef;
|
|
|
4829 }
|
|
|
4830 }
|
|
|
4831 }
|
|
|
4832
|
|
|
4833 ### Bowtie 2 | SINGLE-END | FASTA
|
|
|
4834 sub single_end_align_fragments_to_bisulfite_genome_fastA_bowtie2 {
|
|
|
4835 my ($C_to_T_infile,$G_to_A_infile) = @_;
|
|
|
4836 if ($directional){
|
|
|
4837 print "Input file is $C_to_T_infile (FastA)\n";
|
|
|
4838 }
|
|
|
4839 else{
|
|
|
4840 print "Input files are $C_to_T_infile and $G_to_A_infile (FastA)\n";
|
|
|
4841 }
|
|
|
4842
|
|
|
4843 ## Now starting up to 4 instances of Bowtie feeding in the converted sequence files and reading in the first line of the bowtie output, and storing it in
|
|
|
4844 ## data structure above
|
|
|
4845 if ($directional){
|
|
|
4846 warn "Now running 2 instances of Bowtie 2 against the bisulfite genome of $genome_folder with the specified options: $bowtie_options\n\n";
|
|
|
4847 }
|
|
|
4848 else{
|
|
|
4849 warn "Now running 4 individual instances of Bowtie 2 against the bisulfite genome of $genome_folder with the specified options: $bowtie_options\n\n";
|
|
|
4850 }
|
|
|
4851
|
|
|
4852 foreach my $fh (@fhs) {
|
|
|
4853
|
|
|
4854 my $bt2_options = $bowtie_options;
|
|
|
4855 if ($fh->{name} eq 'CTreadCTgenome' or $fh->{name} eq 'GAreadGAgenome'){
|
|
|
4856 $bt2_options .= ' --norc'; ### ensuring the alignments are only reported in a sensible manner
|
|
|
4857 }
|
|
|
4858 else {
|
|
|
4859 $bt2_options .= ' --nofw';
|
|
|
4860 }
|
|
|
4861
|
|
|
4862 warn "Now starting the Bowtie 2 aligner for $fh->{name} (reading in sequences from $temp_dir$fh->{inputfile} with options: $bt2_options)\n";
|
|
|
4863 open ($fh->{fh},"$path_to_bowtie $bt2_options $fh->{bisulfiteIndex} -U $temp_dir$fh->{inputfile} |") or die "Can't open pipe to bowtie: $!";
|
|
|
4864
|
|
|
4865 ### Bowtie 2 outputs out SAM format, so we need to skip everything until the first sequence
|
|
|
4866 while (1){
|
|
|
4867 $_ = $fh->{fh}->getline();
|
|
|
4868 if ($_) {
|
|
|
4869 last unless ($_ =~ /^\@/); # SAM headers start with @
|
|
|
4870 }
|
|
|
4871 else{
|
|
|
4872 last; # no alignment output
|
|
|
4873 }
|
|
|
4874 }
|
|
|
4875
|
|
|
4876 # Bowtie 2 outputs a result line even for sequences without any alignments. We thus store the first line of the output
|
|
|
4877 if ($_) {
|
|
|
4878 chomp;
|
|
|
4879 my $id = (split(/\t/))[0]; # this is the first element of the Bowtie output (= the sequence identifier)
|
|
|
4880 $fh->{last_seq_id} = $id;
|
|
|
4881 $fh->{last_line} = $_;
|
|
|
4882 warn "Found first alignment:\t$fh->{last_line}\n";
|
|
|
4883 }
|
|
|
4884 # 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
|
|
|
4885 else {
|
|
|
4886 print "Found no alignment, assigning undef to last_seq_id and last_line\n";
|
|
|
4887 $fh->{last_seq_id} = undef;
|
|
|
4888 $fh->{last_line} = undef;
|
|
|
4889 }
|
|
|
4890 }
|
|
|
4891 }
|
|
|
4892
|
|
|
4893
|
|
|
4894 ### Bowtie 1 (default) | SINGLE-END | FASTQ
|
|
|
4895 sub single_end_align_fragments_to_bisulfite_genome_fastQ {
|
|
|
4896 my ($C_to_T_infile,$G_to_A_infile) = @_;
|
|
|
4897 if ($directional){
|
|
|
4898 print "Input file is $C_to_T_infile (FastQ)\n";
|
|
|
4899 }
|
|
|
4900 else{
|
|
|
4901 print "Input files are $C_to_T_infile and $G_to_A_infile (FastQ)\n";
|
|
|
4902 }
|
|
|
4903
|
|
|
4904 ## Now starting up to 4 instances of Bowtie feeding in the converted sequence files and reading in the first line of the bowtie output, and storing it in
|
|
|
4905 ## the data structure above
|
|
|
4906 if ($directional){
|
|
|
4907 warn "Now running 2 instances of Bowtie against the bisulfite genome of $genome_folder with the specified options: $bowtie_options\n\n";
|
|
|
4908 }
|
|
|
4909 else{
|
|
|
4910 warn "Now running 4 individual instances of Bowtie against the bisulfite genome of $genome_folder with the specified options: $bowtie_options\n\n";
|
|
|
4911 }
|
|
|
4912
|
|
|
4913 foreach my $fh (@fhs) {
|
|
|
4914 my $bt_options = $bowtie_options;
|
|
|
4915 if ($fh->{name} eq 'CTreadCTgenome' or $fh->{name} eq 'GAreadGAgenome'){
|
|
|
4916 $bt_options .= ' --norc'; ### ensuring the alignments are only reported in a sensible manner
|
|
|
4917 }
|
|
|
4918 else {
|
|
|
4919 $bt_options .= ' --nofw';
|
|
|
4920 }
|
|
|
4921
|
|
|
4922 warn "Now starting the Bowtie aligner for $fh->{name} (reading in sequences from $temp_dir$fh->{inputfile} with options: $bt_options)\n";
|
|
|
4923 open ($fh->{fh},"$path_to_bowtie $bowtie_options $fh->{bisulfiteIndex} $temp_dir$fh->{inputfile} |") or die "Can't open pipe to bowtie: $!";
|
|
|
4924
|
|
|
4925 # if Bowtie produces an alignment we store the first line of the output
|
|
|
4926 $_ = $fh->{fh}->getline();
|
|
|
4927 if ($_) {
|
|
|
4928 chomp;
|
|
|
4929 my $id = (split(/\t/))[0]; # this is the first element of the Bowtie output (= the sequence identifier)
|
|
|
4930 $fh->{last_seq_id} = $id;
|
|
|
4931 $fh->{last_line} = $_;
|
|
|
4932 warn "Found first alignment:\t$fh->{last_line}\n";
|
|
|
4933 }
|
|
|
4934 # otherwise we just initialise last_seq_id and last_line as undefined
|
|
|
4935 else {
|
|
|
4936 print "Found no alignment, assigning undef to last_seq_id and last_line\n";
|
|
|
4937 $fh->{last_seq_id} = undef;
|
|
|
4938 $fh->{last_line} = undef;
|
|
|
4939 }
|
|
|
4940 }
|
|
|
4941 }
|
|
|
4942
|
|
|
4943 ### Bowtie 2 | SINGLE-END | FASTQ
|
|
|
4944 sub single_end_align_fragments_to_bisulfite_genome_fastQ_bowtie2 {
|
|
|
4945 my ($C_to_T_infile,$G_to_A_infile) = @_;
|
|
|
4946 if ($directional){
|
|
|
4947 print "Input file is $C_to_T_infile (FastQ)\n\n";
|
|
|
4948 }
|
|
|
4949 else{
|
|
|
4950 print "Input files are $C_to_T_infile and $G_to_A_infile (FastQ)\n\n";
|
|
|
4951 }
|
|
|
4952
|
|
|
4953 ## Now starting up to 4 instances of Bowtie 2 feeding in the converted sequence files and reading in the first line of the bowtie output, and storing it in
|
|
|
4954 ## the data structure above
|
|
|
4955 if ($directional){
|
|
|
4956 warn "Now running 2 instances of Bowtie 2 against the bisulfite genome of $genome_folder with the specified options: $bowtie_options\n\n";
|
|
|
4957 }
|
|
|
4958 else{
|
|
|
4959 warn "Now running 4 individual instances of Bowtie 2 against the bisulfite genome of $genome_folder with the specified options: $bowtie_options\n\n";
|
|
|
4960 }
|
|
|
4961
|
|
|
4962 foreach my $fh (@fhs) {
|
|
|
4963 my $bt2_options = $bowtie_options;
|
|
|
4964 if ($fh->{name} eq 'CTreadCTgenome' or $fh->{name} eq 'GAreadGAgenome'){
|
|
|
4965 $bt2_options .= ' --norc'; ### ensuring the alignments are only reported in a sensible manner
|
|
|
4966 }
|
|
|
4967 else {
|
|
|
4968 $bt2_options .= ' --nofw';
|
|
|
4969 }
|
|
|
4970 warn "Now starting the Bowtie 2 aligner for $fh->{name} (reading in sequences from $temp_dir$fh->{inputfile} with options $bt2_options)\n";
|
|
|
4971 warn "Using Bowtie 2 index: $fh->{bisulfiteIndex}\n\n";
|
|
|
4972
|
|
|
4973 open ($fh->{fh},"$path_to_bowtie $bt2_options $fh->{bisulfiteIndex} -U $temp_dir$fh->{inputfile} |") or die "Can't open pipe to bowtie: $!";
|
|
|
4974 ### Bowtie 2 outputs out SAM format, so we need to skip everything until the first sequence
|
|
|
4975 while (1){
|
|
|
4976 $_ = $fh->{fh}->getline();
|
|
|
4977 if ($_) {
|
|
|
4978 last unless ($_ =~ /^\@/); # SAM headers start with @
|
|
|
4979 }
|
|
|
4980 else {
|
|
|
4981 last;
|
|
|
4982 }
|
|
|
4983 }
|
|
|
4984
|
|
|
4985 # Bowtie 2 outputs a result line even for sequences without any alignments. We thus store the first line of the output
|
|
|
4986 if ($_) {
|
|
|
4987 chomp;
|
|
|
4988 my $id = (split(/\t/))[0]; # this is the first element of the Bowtie 2 output (= the sequence identifier)
|
|
|
4989 $fh->{last_seq_id} = $id;
|
|
|
4990 $fh->{last_line} = $_;
|
|
|
4991 warn "Found first alignment:\t$fh->{last_line}\n";
|
|
|
4992 }
|
|
|
4993 # 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
|
|
|
4994 else {
|
|
|
4995 print "Found no alignment, assigning undef to last_seq_id and last_line\n";
|
|
|
4996 $fh->{last_seq_id} = undef;
|
|
|
4997 $fh->{last_line} = undef;
|
|
|
4998 }
|
|
|
4999 }
|
|
|
5000 }
|
|
|
5001
|
|
|
5002 ###########################################################################################################################################
|
|
|
5003
|
|
|
5004 sub reset_counters_and_fhs{
|
|
|
5005 my $filename = shift;
|
|
|
5006 %counting=(
|
|
|
5007 total_meCHH_count => 0,
|
|
|
5008 total_meCHG_count => 0,
|
|
|
5009 total_meCpG_count => 0,
|
|
|
5010 total_unmethylated_CHH_count => 0,
|
|
|
5011 total_unmethylated_CHG_count => 0,
|
|
|
5012 total_unmethylated_CpG_count => 0,
|
|
|
5013 sequences_count => 0,
|
|
|
5014 no_single_alignment_found => 0,
|
|
|
5015 unsuitable_sequence_count => 0,
|
|
|
5016 genomic_sequence_could_not_be_extracted_count => 0,
|
|
|
5017 unique_best_alignment_count => 0,
|
|
|
5018 low_complexity_alignments_overruled_count => 0,
|
|
|
5019 CT_CT_count => 0, #(CT read/CT genome, original top strand)
|
|
|
5020 CT_GA_count => 0, #(CT read/GA genome, original bottom strand)
|
|
|
5021 GA_CT_count => 0, #(GA read/CT genome, complementary to original top strand)
|
|
|
5022 GA_GA_count => 0, #(GA read/GA genome, complementary to original bottom strand)
|
|
|
5023 CT_GA_CT_count => 0, #(CT read1/GA read2/CT genome, original top strand)
|
|
|
5024 GA_CT_GA_count => 0, #(GA read1/CT read2/GA genome, complementary to original bottom strand)
|
|
|
5025 GA_CT_CT_count => 0, #(GA read1/CT read2/CT genome, complementary to original top strand)
|
|
|
5026 CT_GA_GA_count => 0, #(CT read1/GA read2/GA genome, original bottom strand)
|
|
|
5027 alignments_rejected_count => 0, # only relevant if --directional was specified
|
|
|
5028 );
|
|
|
5029
|
|
|
5030 if ($directional){
|
|
|
5031 if ($filename =~ ','){ # paired-end files
|
|
|
5032 @fhs=(
|
|
|
5033 { name => 'CTreadCTgenome',
|
|
|
5034 strand_identity => 'con ori forward',
|
|
|
5035 bisulfiteIndex => $CT_index_basename,
|
|
|
5036 seen => 0,
|
|
|
5037 wrong_strand => 0,
|
|
|
5038 },
|
|
|
5039 { name => 'CTreadGAgenome',
|
|
|
5040 strand_identity => 'con ori reverse',
|
|
|
5041 bisulfiteIndex => $GA_index_basename,
|
|
|
5042 seen => 0,
|
|
|
5043 wrong_strand => 0,
|
|
|
5044 },
|
|
|
5045 { name => 'GAreadCTgenome',
|
|
|
5046 strand_identity => 'compl ori con forward',
|
|
|
5047 bisulfiteIndex => $CT_index_basename,
|
|
|
5048 seen => 0,
|
|
|
5049 wrong_strand => 0,
|
|
|
5050 },
|
|
|
5051 { name => 'GAreadGAgenome',
|
|
|
5052 strand_identity => 'compl ori con reverse',
|
|
|
5053 bisulfiteIndex => $GA_index_basename,
|
|
|
5054 seen => 0,
|
|
|
5055 wrong_strand => 0,
|
|
|
5056 },
|
|
|
5057 );
|
|
|
5058 }
|
|
|
5059 else{ # single-end files
|
|
|
5060 @fhs=(
|
|
|
5061 { name => 'CTreadCTgenome',
|
|
|
5062 strand_identity => 'con ori forward',
|
|
|
5063 bisulfiteIndex => $CT_index_basename,
|
|
|
5064 seen => 0,
|
|
|
5065 wrong_strand => 0,
|
|
|
5066 },
|
|
|
5067 { name => 'CTreadGAgenome',
|
|
|
5068 strand_identity => 'con ori reverse',
|
|
|
5069 bisulfiteIndex => $GA_index_basename,
|
|
|
5070 seen => 0,
|
|
|
5071 wrong_strand => 0,
|
|
|
5072 },
|
|
|
5073 );
|
|
|
5074 }
|
|
|
5075 }
|
|
|
5076 else{
|
|
|
5077 @fhs=(
|
|
|
5078 { name => 'CTreadCTgenome',
|
|
|
5079 strand_identity => 'con ori forward',
|
|
|
5080 bisulfiteIndex => $CT_index_basename,
|
|
|
5081 seen => 0,
|
|
|
5082 wrong_strand => 0,
|
|
|
5083 },
|
|
|
5084 { name => 'CTreadGAgenome',
|
|
|
5085 strand_identity => 'con ori reverse',
|
|
|
5086 bisulfiteIndex => $GA_index_basename,
|
|
|
5087 seen => 0,
|
|
|
5088 wrong_strand => 0,
|
|
|
5089 },
|
|
|
5090 { name => 'GAreadCTgenome',
|
|
|
5091 strand_identity => 'compl ori con forward',
|
|
|
5092 bisulfiteIndex => $CT_index_basename,
|
|
|
5093 seen => 0,
|
|
|
5094 wrong_strand => 0,
|
|
|
5095 },
|
|
|
5096 { name => 'GAreadGAgenome',
|
|
|
5097 strand_identity => 'compl ori con reverse',
|
|
|
5098 bisulfiteIndex => $GA_index_basename,
|
|
|
5099 seen => 0,
|
|
|
5100 wrong_strand => 0,
|
|
|
5101 },
|
|
|
5102 );
|
|
|
5103 }
|
|
|
5104 }
|
|
|
5105
|
|
|
5106
|
|
|
5107 sub process_command_line{
|
|
|
5108 my @bowtie_options;
|
|
|
5109 my $help;
|
|
|
5110 my $mates1;
|
|
|
5111 my $mates2;
|
|
|
5112 my $path_to_bowtie;
|
|
|
5113 my $fastq;
|
|
|
5114 my $fasta;
|
|
|
5115 my $skip;
|
|
|
5116 my $qupto;
|
|
|
5117 my $phred64;
|
|
|
5118 my $phred33;
|
|
|
5119 my $solexa;
|
|
|
5120 my $mismatches;
|
|
|
5121 my $seed_length;
|
|
|
5122 my $best;
|
|
|
5123 my $sequence_format;
|
|
|
5124 my $version;
|
|
|
5125 my $quiet;
|
|
|
5126 my $chunk;
|
|
|
5127 my $non_directional;
|
|
|
5128 my $ceiling;
|
|
|
5129 my $maxins;
|
|
|
5130 my $minins;
|
|
|
5131 my $unmapped;
|
|
|
5132 my $multi_map;
|
|
|
5133 my $output_dir;
|
|
|
5134 my $bowtie2;
|
|
|
5135 my $vanilla;
|
|
|
5136 my $sam_no_hd;
|
|
|
5137 my $seed_extension_fails;
|
|
|
5138 my $reseed_repetitive_seeds;
|
|
|
5139 my $most_valid_alignments;
|
|
|
5140 my $score_min;
|
|
|
5141 my $parallel;
|
|
|
5142 my $temp_dir;
|
|
|
5143
|
|
|
5144 my $command_line = GetOptions ('help|man' => \$help,
|
|
|
5145 '1=s' => \$mates1,
|
|
|
5146 '2=s' => \$mates2,
|
|
|
5147 'path_to_bowtie=s' => \$path_to_bowtie,
|
|
|
5148 'f|fasta' => \$fasta,
|
|
|
5149 'q|fastq' => \$fastq,
|
|
|
5150 's|skip=i' => \$skip,
|
|
|
5151 'u|upto=i' => \$qupto,
|
|
|
5152 'phred33-quals' => \$phred33,
|
|
|
5153 'phred64-quals|solexa1' => \$phred64,
|
|
|
5154 'solexa-quals' => \$solexa,
|
|
|
5155 'n|seedmms=i' => \$mismatches,
|
|
|
5156 'l|seedlen=i' => \$seed_length,
|
|
|
5157 'no_best' => \$best,
|
|
|
5158 'version' => \$version,
|
|
|
5159 'quiet' => \$quiet,
|
|
|
5160 'chunkmbs=i' => \$chunk,
|
|
|
5161 'non_directional' => \$non_directional,
|
|
|
5162 'I|minins=i' => \$minins,
|
|
|
5163 'X|maxins=i' => \$maxins,
|
|
|
5164 'e|maqerr=i' => \$ceiling,
|
|
|
5165 'un|unmapped' => \$unmapped,
|
|
|
5166 'ambiguous' => \$multi_map,
|
|
|
5167 'o|output_dir=s' => \$output_dir,
|
|
|
5168 'bowtie2' => \$bowtie2,
|
|
|
5169 'vanilla' => \$vanilla,
|
|
|
5170 'sam-no-hd' => \$sam_no_hd,
|
|
|
5171 'D=i' => \$seed_extension_fails,
|
|
|
5172 'R=i' => \$reseed_repetitive_seeds,
|
|
|
5173 'score_min=s' => \$score_min,
|
|
|
5174 'most_valid_alignments=i' => \$most_valid_alignments,
|
|
|
5175 'p=i' => \$parallel,
|
|
|
5176 'temp_dir=s' => \$temp_dir,
|
|
|
5177 );
|
|
|
5178
|
|
|
5179
|
|
|
5180 ### EXIT ON ERROR if there were errors with any of the supplied options
|
|
|
5181 unless ($command_line){
|
|
|
5182 die "Please respecify command line options\n";
|
|
|
5183 }
|
|
|
5184 ### HELPFILE
|
|
|
5185 if ($help){
|
|
|
5186 print_helpfile();
|
|
|
5187 exit;
|
|
|
5188 }
|
|
|
5189 if ($version){
|
|
|
5190 print << "VERSION";
|
|
|
5191
|
|
|
5192
|
|
|
5193 Bismark - Bisulfite Mapper and Methylation Caller.
|
|
|
5194
|
|
|
5195 Bismark Version: $bismark_version Copyright 2010-12 Felix Krueger, Babraham Bioinformatics
|
|
|
5196 www.bioinformatics.babraham.ac.uk/projects/
|
|
|
5197
|
|
|
5198
|
|
|
5199 VERSION
|
|
|
5200 exit;
|
|
|
5201 }
|
|
|
5202
|
|
|
5203
|
|
|
5204 ##########################
|
|
|
5205 ### PROCESSING OPTIONS ###
|
|
|
5206 ##########################
|
|
|
5207
|
|
|
5208 unless ($bowtie2){
|
|
|
5209 $bowtie2 = 0;
|
|
|
5210 }
|
|
|
5211 unless ($sam_no_hd){
|
|
|
5212 $sam_no_hd =0;
|
|
|
5213 }
|
|
|
5214
|
|
|
5215 ### PATH TO BOWTIE
|
|
|
5216 ### if a special path to Bowtie 1/2 was specified we will use that one, otherwise it is assumed that Bowtie 1/2 is in the PATH
|
|
|
5217 if ($path_to_bowtie){
|
|
|
5218 unless ($path_to_bowtie =~ /\/$/){
|
|
|
5219 $path_to_bowtie =~ s/$/\//;
|
|
|
5220 }
|
|
|
5221 if (-d $path_to_bowtie){
|
|
|
5222 if ($bowtie2){
|
|
|
5223 $path_to_bowtie = "${path_to_bowtie}bowtie2";
|
|
|
5224 }
|
|
|
5225 else{
|
|
|
5226 $path_to_bowtie = "${path_to_bowtie}bowtie";
|
|
|
5227 }
|
|
|
5228 }
|
|
|
5229 else{
|
|
|
5230 die "The path to bowtie provided ($path_to_bowtie) is invalid (not a directory)!\n";
|
|
|
5231 }
|
|
|
5232 }
|
|
|
5233 else{
|
|
|
5234 if ($bowtie2){
|
|
|
5235 $path_to_bowtie = 'bowtie2';
|
|
|
5236 warn "Path to Bowtie 2 specified as: $path_to_bowtie\n"; }
|
|
|
5237 else{
|
|
|
5238 $path_to_bowtie = 'bowtie';
|
|
|
5239 warn "Path to Bowtie specified as: $path_to_bowtie\n";
|
|
|
5240 }
|
|
|
5241 }
|
|
|
5242
|
|
|
5243 ####################################
|
|
|
5244 ### PROCESSING ARGUMENTS
|
|
|
5245
|
|
|
5246 ### GENOME FOLDER
|
|
|
5247 my $genome_folder = shift @ARGV; # mandatory
|
|
|
5248 unless ($genome_folder){
|
|
|
5249 warn "Genome folder was not specified!\n";
|
|
|
5250 print_helpfile();
|
|
|
5251 exit;
|
|
|
5252 }
|
|
|
5253
|
|
|
5254 ### checking that the genome folder, all subfolders and the required bowtie index files exist
|
|
|
5255 unless ($genome_folder =~/\/$/){
|
|
|
5256 $genome_folder =~ s/$/\//;
|
|
|
5257 }
|
|
|
5258
|
|
|
5259 if (chdir $genome_folder){
|
|
|
5260 my $absolute_genome_folder = getcwd; ## making the genome folder path absolute
|
|
|
5261 unless ($absolute_genome_folder =~/\/$/){
|
|
|
5262 $absolute_genome_folder =~ s/$/\//;
|
|
|
5263 }
|
|
|
5264 warn "Reference genome folder provided is $genome_folder\t(absolute path is '$absolute_genome_folder)'\n";
|
|
|
5265 $genome_folder = $absolute_genome_folder;
|
|
|
5266 }
|
|
|
5267 else{
|
|
|
5268 die "Failed to move to $genome_folder: $!\nUSAGE: Bismark.pl [options] <genome_folder> {-1 <mates1> -2 <mates2> | <singles>} [<hits>] (--help for more details)\n";
|
|
|
5269 }
|
|
|
5270
|
|
|
5271 my $CT_dir = "${genome_folder}Bisulfite_Genome/CT_conversion/";
|
|
|
5272 my $GA_dir = "${genome_folder}Bisulfite_Genome/GA_conversion/";
|
|
|
5273
|
|
|
5274 if ($bowtie2){ ### Bowtie 2 (new)
|
|
|
5275 ### checking the integrity of $CT_dir
|
|
|
5276 chdir $CT_dir or die "Failed to move to directory $CT_dir: $!\n";
|
|
|
5277 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');
|
|
|
5278 foreach my $file(@CT_bowtie_index){
|
|
|
5279 unless (-f $file){
|
|
|
5280 die "The Bowtie 2 index of the C->T converted genome seems to be faulty ($file). Please run the bismark_genome_preparation before running Bismark.\n";
|
|
|
5281 }
|
|
|
5282 }
|
|
|
5283 ### checking the integrity of $GA_dir
|
|
|
5284 chdir $GA_dir or die "Failed to move to directory $GA_dir: $!\n";
|
|
|
5285 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');
|
|
|
5286 foreach my $file(@GA_bowtie_index){
|
|
|
5287 unless (-f $file){
|
|
|
5288 die "The Bowtie 2 index of the G->A converted genome seems to be faulty ($file). Please run bismark_genome_preparation before running Bismark.\n";
|
|
|
5289 }
|
|
|
5290 }
|
|
|
5291 }
|
|
|
5292
|
|
|
5293 else{ ### Bowtie 1 (default)
|
|
|
5294 ### checking the integrity of $CT_dir
|
|
|
5295 chdir $CT_dir or die "Failed to move to directory $CT_dir: $!\n";
|
|
|
5296 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');
|
|
|
5297 foreach my $file(@CT_bowtie_index){
|
|
|
5298 unless (-f $file){
|
|
|
5299 die "The Bowtie index of the C->T converted genome seems to be faulty ($file). Please run bismark_genome_preparation before running Bismark.\n";
|
|
|
5300 }
|
|
|
5301 }
|
|
|
5302 ### checking the integrity of $GA_dir
|
|
|
5303 chdir $GA_dir or die "Failed to move to directory $GA_dir: $!\n";
|
|
|
5304 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');
|
|
|
5305 foreach my $file(@GA_bowtie_index){
|
|
|
5306 unless (-f $file){
|
|
|
5307 die "The Bowtie index of the G->A converted genome seems to be faulty ($file). Please run bismark_genome_preparation before running Bismark.\n";
|
|
|
5308 }
|
|
|
5309 }
|
|
|
5310 }
|
|
|
5311
|
|
|
5312 my $CT_index_basename = "${CT_dir}BS_CT";
|
|
|
5313 my $GA_index_basename = "${GA_dir}BS_GA";
|
|
|
5314
|
|
|
5315 ### INPUT OPTIONS
|
|
|
5316
|
|
|
5317 ### SEQUENCE FILE FORMAT
|
|
|
5318 ### exits if both fastA and FastQ were specified
|
|
|
5319 if ($fasta and $fastq){
|
|
|
5320 die "Only one sequence filetype can be specified (fastA or fastQ)\n";
|
|
|
5321 }
|
|
|
5322
|
|
|
5323 ### unless fastA is specified explicitely, fastQ sequence format is expected by default
|
|
|
5324 if ($fasta){
|
|
|
5325 print "FastA format specified\n";
|
|
|
5326 $sequence_format = 'FASTA';
|
|
|
5327 push @bowtie_options, '-f';
|
|
|
5328 }
|
|
|
5329 elsif ($fastq){
|
|
|
5330 print "FastQ format specified\n";
|
|
|
5331 $sequence_format = 'FASTQ';
|
|
|
5332 push @bowtie_options, '-q';
|
|
|
5333 }
|
|
|
5334 else{
|
|
|
5335 $fastq = 1;
|
|
|
5336 print "FastQ format assumed (by default)\n";
|
|
|
5337 $sequence_format = 'FASTQ';
|
|
|
5338 push @bowtie_options, '-q';
|
|
|
5339 }
|
|
|
5340
|
|
|
5341 ### SKIP
|
|
|
5342 if ($skip){
|
|
|
5343 warn "Skipping the first $skip reads from the input file\n";
|
|
|
5344 # push @bowtie_options,"-s $skip";
|
|
|
5345 }
|
|
|
5346
|
|
|
5347 ### UPTO
|
|
|
5348 if ($qupto){
|
|
|
5349 warn "Processing sequences up to read no. $qupto from the input file\n";
|
|
|
5350 if ($bowtie2){
|
|
|
5351 # push @bowtie_options,"--upto $qupto"; ## slightly changed for Bowtie 2
|
|
|
5352 }
|
|
|
5353 else{
|
|
|
5354 # push @bowtie_options,"--qupto $qupto";
|
|
|
5355 }
|
|
|
5356 }
|
|
|
5357
|
|
|
5358 ### QUALITY VALUES
|
|
|
5359 if (($phred33 and $phred64) or ($phred33 and $solexa) or ($phred64 and $solexa)){
|
|
|
5360 die "You can only specify one type of quality value at a time! (--phred33-quals or --phred64-quals or --solexa-quals)";
|
|
|
5361 }
|
|
|
5362 if ($phred33){ ## if nothing else is specified $phred33 will be used as default by both Bowtie 1 and 2.
|
|
|
5363 # Phred quality values work only when -q is specified
|
|
|
5364 unless ($fastq){
|
|
|
5365 die "Phred quality values works only when -q (FASTQ) is specified\n";
|
|
|
5366 }
|
|
|
5367 if ($bowtie2){
|
|
|
5368 push @bowtie_options,"--phred33";
|
|
|
5369 }
|
|
|
5370 else{
|
|
|
5371 push @bowtie_options,"--phred33-quals";
|
|
|
5372 }
|
|
|
5373 }
|
|
|
5374 if ($phred64){
|
|
|
5375 # Phred quality values work only when -q is specified
|
|
|
5376 unless ($fastq){
|
|
|
5377 die "Phred quality values work only when -q (FASTQ) is specified\n";
|
|
|
5378 }
|
|
|
5379 if ($bowtie2){
|
|
|
5380 push @bowtie_options,"--phred64";
|
|
|
5381 }
|
|
|
5382 else{
|
|
|
5383 push @bowtie_options,"--phred64-quals";
|
|
|
5384 }
|
|
|
5385 }
|
|
|
5386 else{
|
|
|
5387 $phred64 = 0;
|
|
|
5388 }
|
|
|
5389
|
|
|
5390 if ($solexa){
|
|
|
5391 if ($bowtie2){
|
|
|
5392 die "The option '--solexa-quals' is not compatible with Bowtie 2. Please respecify!\n";
|
|
|
5393 }
|
|
|
5394 # Solexa to Phred value conversion works only when -q is specified
|
|
|
5395 unless ($fastq){
|
|
|
5396 die "Conversion from Solexa to Phred quality values works only when -q (FASTQ) is specified\n";
|
|
|
5397 }
|
|
|
5398 push @bowtie_options,"--solexa-quals";
|
|
|
5399 }
|
|
|
5400 else{
|
|
|
5401 $solexa = 0;
|
|
|
5402 }
|
|
|
5403
|
|
|
5404 ### ALIGNMENT OPTIONS
|
|
|
5405
|
|
|
5406 ### MISMATCHES
|
|
|
5407 if (defined $mismatches){
|
|
|
5408 if ($bowtie2){
|
|
|
5409 if ($mismatches == 0 or $mismatches == 1){
|
|
|
5410 push @bowtie_options,"-N $mismatches";
|
|
|
5411 }
|
|
|
5412 else{
|
|
|
5413 die "Please set the number of multiseed mismatches for Bowtie 2 with '-N <int>' (where <int> can be 0 or 1)\n";
|
|
|
5414 }
|
|
|
5415 }
|
|
|
5416 else{
|
|
|
5417 if ($mismatches >= 0 and $mismatches <= 3){
|
|
|
5418 push @bowtie_options,"-n $mismatches";
|
|
|
5419 }
|
|
|
5420 else{
|
|
|
5421 die "Please set the number of seed mismatches for Bowtie 1 with '-n <int>' (where <int> can be 0,1,2 or 3)\n";
|
|
|
5422 }
|
|
|
5423 }
|
|
|
5424 }
|
|
|
5425 else{
|
|
|
5426 unless ($bowtie2){
|
|
|
5427 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
|
|
|
5428 }
|
|
|
5429 }
|
|
|
5430
|
|
|
5431 ### SEED LENGTH
|
|
|
5432 if (defined $seed_length){
|
|
|
5433 if ($bowtie2){
|
|
|
5434 push @bowtie_options,"-L $seed_length";
|
|
|
5435 }
|
|
|
5436 else{
|
|
|
5437 push @bowtie_options,"-l $seed_length";
|
|
|
5438 }
|
|
|
5439 }
|
|
|
5440
|
|
|
5441 ### MISMATCH CEILING
|
|
|
5442 if (defined $ceiling){
|
|
|
5443 die "The option '-e' is not compatible with Bowtie 2. Please respecify options\n" if ($bowtie2);
|
|
|
5444 push @bowtie_options,"-e $ceiling";
|
|
|
5445 }
|
|
|
5446
|
|
|
5447
|
|
|
5448 ### BOWTIE 2 EFFORT OPTIONS
|
|
|
5449
|
|
|
5450 ### CONSECUTIVE SEED EXTENSION FAILS
|
|
|
5451 if (defined $seed_extension_fails){
|
|
|
5452 die "The option '-D <int>' is only available when using Bowtie 2\n\n" unless ($bowtie2);
|
|
|
5453 push @bowtie_options,"-D $seed_extension_fails";
|
|
|
5454 }
|
|
|
5455
|
|
|
5456 ### RE-SEEDING REPETITIVE SEEDS
|
|
|
5457 if (defined $reseed_repetitive_seeds){
|
|
|
5458 die "The option '-R <int>' is only available when using Bowtie 2\n\n" unless ($bowtie2);
|
|
|
5459 push @bowtie_options,"-R $reseed_repetitive_seeds";
|
|
|
5460 }
|
|
|
5461
|
|
|
5462
|
|
|
5463 ### BOWTIE 2 SCORING OPTIONS
|
|
|
5464 if ($score_min){
|
|
|
5465 die "The option '--score_min <func>' is only available when using Bowtie 2\n\n" unless ($bowtie2);
|
|
|
5466 unless ($score_min =~ /^L,.+,.+$/){
|
|
|
5467 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";
|
|
|
5468 }
|
|
|
5469 push @bowtie_options,"--score-min $score_min";
|
|
|
5470 }
|
|
|
5471 else{
|
|
|
5472 if ($bowtie2){
|
|
|
5473 push @bowtie_options,"--score-min L,0,-0.2"; # default setting, more stringent than normal Bowtie2
|
|
|
5474 }
|
|
|
5475 }
|
|
|
5476
|
|
|
5477 ### BOWTIE 2 PARALLELIZATION OPTIONS
|
|
|
5478 if (defined $parallel){
|
|
|
5479 die "The parallelization switch '-p' only works for Bowtie 2. Please respecify!" unless ($bowtie2);
|
|
|
5480 }
|
|
|
5481 if ($bowtie2){
|
|
|
5482 if ($parallel){
|
|
|
5483 die "Please select a value for -p of 2 or more!\n" unless ($parallel > 1);
|
|
|
5484 push @bowtie_options,"-p $parallel";
|
|
|
5485 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.
|
|
|
5486 print "Each Bowtie 2 instance is going to be run with $parallel threads. Please monitor performance closely and tune down if needed!\n";
|
|
|
5487 sleep (2);
|
|
|
5488 }
|
|
|
5489 }
|
|
|
5490
|
|
|
5491 ### REPORTING OPTIONS
|
|
|
5492
|
|
|
5493 if ($bowtie2){
|
|
|
5494 push @bowtie_options,'--ignore-quals'; ## All mismatches will receive penalty for mismatches as if they were of high quality, which is 6 by default
|
|
|
5495
|
|
|
5496 ### Option -M is deprecated since Bowtie 2 version 2.0.0 beta7. I'll leave this option commented out for a while
|
|
|
5497 if(defined $most_valid_alignments){
|
|
|
5498
|
|
|
5499 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";
|
|
|
5500 # push @bowtie_options,"-M $most_valid_alignments";sleep (5);
|
|
|
5501 }
|
|
|
5502 # else{
|
|
|
5503 # push @bowtie_options,'-M 10'; # the default behavior for Bowtie 2 is to report (and sort) up to 500 alignments for a given sequence
|
|
|
5504 # }
|
|
|
5505 }
|
|
|
5506 else{ # Because of the way Bismark works we will always use the reporting option -k 2 (report up to 2 valid alignments) for Bowtie 1
|
|
|
5507 push @bowtie_options,'-k 2';
|
|
|
5508 }
|
|
|
5509
|
|
|
5510 ### --BEST
|
|
|
5511 if ($bowtie2){
|
|
|
5512 if ($best){ # Bowtie 2 does away with the concept of --best, so one can also not select --no-best when Bowtie 2 is to be used
|
|
|
5513 die "The option '--no-best' is not compatible with Bowtie 2. Please respecify options\n";
|
|
|
5514 }
|
|
|
5515 }
|
|
|
5516 else{
|
|
|
5517 # --best is the default option for Bowtie 1, specifying --no-best can turn it off (e.g. to speed up alignment process)
|
|
|
5518 unless ($best){
|
|
|
5519 push @bowtie_options,'--best';
|
|
|
5520 }
|
|
|
5521 }
|
|
|
5522
|
|
|
5523 ### VANILLA BISMARK (BOWTIE 1) OUTPUT
|
|
|
5524 if ($vanilla){
|
|
|
5525 if ($bowtie2){
|
|
|
5526 die "The options --bowtie2 and the --vanilla are not compatible. Please respecify!\n\n";
|
|
|
5527 }
|
|
|
5528 }
|
|
|
5529 else{
|
|
|
5530 $vanilla = 0;
|
|
|
5531 }
|
|
|
5532
|
|
|
5533 ### PAIRED-END MAPPING
|
|
|
5534 if ($mates1){
|
|
|
5535 my @mates1 = (split (/,/,$mates1));
|
|
|
5536 die "Paired-end mapping requires the format: -1 <mates1> -2 <mates2>, please respecify!\n" unless ($mates2);
|
|
|
5537 my @mates2 = (split(/,/,$mates2));
|
|
|
5538 unless (scalar @mates1 == scalar @mates2){
|
|
|
5539 die "Paired-end mapping requires the same amounnt of mate1 and mate2 files, please respecify! (format: -1 <mates1> -2 <mates2>)\n";
|
|
|
5540 }
|
|
|
5541 while (1){
|
|
|
5542 my $mate1 = shift @mates1;
|
|
|
5543 my $mate2 = shift @mates2;
|
|
|
5544 last unless ($mate1 and $mate2);
|
|
|
5545 push @filenames,"$mate1,$mate2";
|
|
|
5546 }
|
|
|
5547 if ($bowtie2){
|
|
|
5548 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
|
|
|
5549 push @bowtie_options,'--no-discordant';## By default Bowtie 2 is not looking for discordant alignments if it can't find concordant ones
|
|
|
5550 }
|
|
|
5551 }
|
|
|
5552 elsif ($mates2){
|
|
|
5553 die "Paired-end mapping requires the format: -1 <mates1> -2 <mates2>, please respecify!\n";
|
|
|
5554 }
|
|
|
5555
|
|
|
5556 ### SINGLE-END MAPPING
|
|
|
5557 # Single-end mapping will be performed if no mate pairs for paired-end mapping have been specified
|
|
|
5558 my $singles;
|
|
|
5559 unless ($mates1 and $mates2){
|
|
|
5560 $singles = join (',',@ARGV);
|
|
|
5561 unless ($singles){
|
|
|
5562 die "\nNo filename supplied! Please specify one or more files for single-end Bismark mapping!\n";
|
|
|
5563 }
|
|
|
5564 $singles =~ s/\s/,/g;
|
|
|
5565 @filenames = (split(/,/,$singles));
|
|
|
5566 warn "\nFiles to be analysed:\n";
|
|
|
5567 warn "@filenames\n\n";
|
|
|
5568 sleep (3);
|
|
|
5569 }
|
|
|
5570
|
|
|
5571 ### MININUM INSERT SIZE (PAIRED-END ONLY)
|
|
|
5572 if (defined $minins){
|
|
|
5573 die "-I/--minins can only be used for paired-end mapping!\n\n" if ($singles);
|
|
|
5574 push @bowtie_options,"--minins $minins";
|
|
|
5575 }
|
|
|
5576
|
|
|
5577 ### MAXIMUM INSERT SIZE (PAIRED-END ONLY)
|
|
|
5578 if (defined $maxins){
|
|
|
5579 die "-X/--maxins can only be used for paired-end mapping!\n\n" if ($singles);
|
|
|
5580 push @bowtie_options,"--maxins $maxins";
|
|
|
5581 }
|
|
|
5582 else{
|
|
|
5583 unless ($singles){
|
|
|
5584 push @bowtie_options,'--maxins 500';
|
|
|
5585 }
|
|
|
5586 }
|
|
|
5587
|
|
|
5588 ### QUIET prints nothing besides alignments (suppresses warnings)
|
|
|
5589 if ($quiet){
|
|
|
5590 push @bowtie_options,'--quiet';
|
|
|
5591 }
|
|
|
5592
|
|
|
5593 ### CHUNKMBS needed to be increased to avoid memory exhaustion warnings for Bowtie 1, particularly for --best (and paired-end) alignments
|
|
|
5594 unless ($bowtie2){ # Bowtie 2 does not have a chunkmbs option
|
|
|
5595 if (defined $chunk){
|
|
|
5596 push @bowtie_options,"--chunkmbs $chunk";
|
|
|
5597 }
|
|
|
5598 else{
|
|
|
5599 push @bowtie_options,'--chunkmbs 512'; ## setting the default to 512MB (up from 64 default)
|
|
|
5600 }
|
|
|
5601 }
|
|
|
5602
|
|
|
5603
|
|
|
5604 ### SUMMARY OF ALL BOWTIE OPTIONS
|
|
|
5605 my $bowtie_options = join (' ',@bowtie_options);
|
|
|
5606
|
|
|
5607
|
|
|
5608 ### STRAND-SPECIFIC LIBRARIES
|
|
|
5609 my $directional;
|
|
|
5610 if ($non_directional){
|
|
|
5611 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";
|
|
|
5612 sleep (3);
|
|
|
5613 $directional = 0;
|
|
|
5614 }
|
|
|
5615 else{
|
|
|
5616 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";
|
|
|
5617 sleep (3);
|
|
|
5618 $directional = 1; # Changed this to being the default behaviour
|
|
|
5619 }
|
|
|
5620
|
|
|
5621 ### UNMAPPED SEQUENCE OUTPUT
|
|
|
5622 $unmapped = 0 unless ($unmapped);
|
|
|
5623
|
|
|
5624 ### AMBIGUOUS ALIGNMENT SEQUENCE OUTPUT
|
|
|
5625 $multi_map = 0 unless ($multi_map);
|
|
|
5626
|
|
|
5627
|
|
|
5628 ### OUTPUT DIRECTORY
|
|
|
5629
|
|
|
5630 chdir $parent_dir or die "Failed to move back to current working directory\n";
|
|
|
5631 if ($output_dir){
|
|
|
5632 unless ($output_dir =~ /\/$/){
|
|
|
5633 $output_dir =~ s/$/\//;
|
|
|
5634 }
|
|
|
5635
|
|
|
5636 if (chdir $output_dir){
|
|
|
5637 $output_dir = getcwd; # making the path absolute
|
|
|
5638 unless ($output_dir =~ /\/$/){
|
|
|
5639 $output_dir =~ s/$/\//;
|
|
|
5640 }
|
|
|
5641 }
|
|
|
5642 else{
|
|
|
5643 mkdir $output_dir or die "Unable to create directory $output_dir $!\n";
|
|
|
5644 warn "Created output directory $output_dir!\n\n";
|
|
|
5645 chdir $output_dir or die "Failed to move to $output_dir\n";
|
|
|
5646 $output_dir = getcwd; # making the path absolute
|
|
|
5647 unless ($output_dir =~ /\/$/){
|
|
|
5648 $output_dir =~ s/$/\//;
|
|
|
5649 }
|
|
|
5650 }
|
|
|
5651 warn "Output will be written into the directory: $output_dir\n";
|
|
|
5652 }
|
|
|
5653 else{
|
|
|
5654 $output_dir = '';
|
|
|
5655 }
|
|
|
5656
|
|
|
5657 ### TEMPORARY DIRECTORY for C->T and G->A transcribed files
|
|
|
5658
|
|
|
5659 chdir $parent_dir or die "Failed to move back to current working directory\n";
|
|
|
5660 if ($temp_dir){
|
|
|
5661 warn "\nUsing temp directory: $temp_dir\n";
|
|
|
5662 unless ($temp_dir =~ /\/$/){
|
|
|
5663 $temp_dir =~ s/$/\//;
|
|
|
5664 }
|
|
|
5665
|
|
|
5666 if (chdir $temp_dir){
|
|
|
5667 $temp_dir = getcwd; # making the path absolute
|
|
|
5668 unless ($temp_dir =~ /\/$/){
|
|
|
5669 $temp_dir =~ s/$/\//;
|
|
|
5670 }
|
|
|
5671 }
|
|
|
5672 else{
|
|
|
5673 mkdir $temp_dir or die "Unable to create directory $temp_dir $!\n";
|
|
|
5674 warn "Created temporary directory $temp_dir!\n\n";
|
|
|
5675 chdir $temp_dir or die "Failed to move to $temp_dir\n";
|
|
|
5676 $temp_dir = getcwd; # making the path absolute
|
|
|
5677 unless ($temp_dir =~ /\/$/){
|
|
|
5678 $temp_dir =~ s/$/\//;
|
|
|
5679 }
|
|
|
5680 }
|
|
|
5681 warn "Temporary files will be written into the directory: $temp_dir\n";
|
|
|
5682 }
|
|
|
5683 else{
|
|
|
5684 $temp_dir = '';
|
|
|
5685 }
|
|
|
5686
|
|
|
5687
|
|
|
5688 return ($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);
|
|
|
5689 }
|
|
|
5690
|
|
|
5691
|
|
|
5692
|
|
|
5693 sub generate_SAM_header{
|
|
|
5694 print OUT "\@HD\tVN:1.0\tSO:unsorted\n"; # @HD = header, VN = version, SO = sort order
|
|
|
5695 foreach my $chr (keys %chromosomes){
|
|
|
5696 my $length = length ($chromosomes{$chr});
|
|
|
5697 print OUT "\@SQ\tSN:$chr\tLN:$length\n"; # @SQ = sequence, SN = seq name, LN = length
|
|
|
5698 }
|
|
|
5699 print OUT "\@PG\tID:Bismark\tVN:$bismark_version\tCL:\"bismark $command_line\"\n"; # @PG = program, ID = unique identifier, PN = program name name, VN = program version
|
|
|
5700 }
|
|
|
5701
|
|
|
5702 ### I would like to thank the following individuals for their valuable contributions to the Bismark SAM output format:
|
|
|
5703 ### O. Tam (Sep 2010), C. Whelan (2011), E. Vidal (2011), T. McBryan (2011), P. Hickey (2011)
|
|
|
5704
|
|
|
5705 sub single_end_SAM_output{
|
|
|
5706 my ($id,$actual_seq,$methylation_call_params,$qual) = @_;
|
|
|
5707 my $strand = $methylation_call_params->{$id}->{alignment_strand};
|
|
|
5708 my $chr = $methylation_call_params->{$id}->{chromosome};
|
|
|
5709 my $start = $methylation_call_params->{$id}->{position};
|
|
|
5710 my $stop = $methylation_call_params->{$id}->{end_position};
|
|
|
5711 my $ref_seq = $methylation_call_params->{$id}->{unmodified_genomic_sequence};
|
|
|
5712 my $methcall = $methylation_call_params->{$id}->{methylation_call};
|
|
|
5713 my $read_conversion = $methylation_call_params->{$id}->{read_conversion};
|
|
|
5714 my $genome_conversion = $methylation_call_params->{$id}->{genome_conversion};
|
|
|
5715 my $number_of_mismatches = $methylation_call_params->{$id}->{number_of_mismatches};
|
|
|
5716 ### 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"
|
|
|
5717 ## FLAG: bitwise FLAG. Each bit is explained in the following table:
|
|
|
5718 ## Bit Description Comment Value
|
|
|
5719 ## 0x1 template has multiple segments in sequencing 0: single-end 1: paired end value: 2**0 ( 1)
|
|
|
5720 ## 0x2 each segment properly aligned according to the aligner true only for paired-end alignments value: 2**1 ( 2)
|
|
|
5721 ## 0x4 segment unmapped --- ---
|
|
|
5722 ## 0x8 next segment in the template unmapped --- ---
|
|
|
5723 ## 0x10 SEQ being reverse complemented value: 2**4 ( 16)
|
|
|
5724 ## 0x20 SEQ of the next segment in the template being reversed value: 2**5 ( 32)
|
|
|
5725 ## 0x40 the first segment in the template read 1 value: 2**6 ( 64)
|
|
|
5726 ## 0x80 the last segment in the template read 2 value: 2**7 (128)
|
|
|
5727 ## 0x100 secondary alignment --- ---
|
|
|
5728 ## 0x200 not passing quality controls --- ---
|
|
|
5729 ## 0x400 PCR or optical duplicate --- ---
|
|
|
5730
|
|
|
5731 #####
|
|
|
5732
|
|
|
5733 my $flag; # FLAG variable used for SAM format.
|
|
|
5734 if ($strand eq "+"){
|
|
|
5735 if ($read_conversion eq 'CT' and $genome_conversion eq 'CT'){
|
|
|
5736 $flag = 0; # 0 for "+" strand (OT)
|
|
|
5737 }
|
|
|
5738 elsif ($read_conversion eq 'GA' and $genome_conversion eq 'GA'){
|
|
|
5739 $flag = 16; # 16 for "-" strand (CTOB, yields information for the original bottom strand)
|
|
|
5740 }
|
|
|
5741 else{
|
|
|
5742 die "Unexpected strand and read/genome conversion: strand: $strand, read conversion: $read_conversion, genome_conversion: $genome_conversion\n\n";
|
|
|
5743 }
|
|
|
5744 }
|
|
|
5745 elsif ($strand eq "-"){
|
|
|
5746 if ($read_conversion eq 'CT' and $genome_conversion eq 'GA'){
|
|
|
5747 $flag = 16; # 16 for "-" strand (OB)
|
|
|
5748 }
|
|
|
5749 elsif ($read_conversion eq 'GA' and $genome_conversion eq 'CT'){
|
|
|
5750 $flag = 0; # 0 for "+" strand (CTOT, yields information for the original top strand)
|
|
|
5751 }
|
|
|
5752 else{
|
|
|
5753 die "Unexpected strand and read/genome conversion: strand: $strand, read conversion: $read_conversion, genome_conversion: $genome_conversion\n\n";
|
|
|
5754 }
|
|
|
5755 }
|
|
|
5756 else{
|
|
|
5757 die "Unexpected strand information: $strand\n\n";
|
|
|
5758 }
|
|
|
5759
|
|
|
5760 #####
|
|
|
5761
|
|
|
5762 my $mapq = 255; # Assume mapping quality is unavailable
|
|
|
5763
|
|
|
5764 #####
|
|
|
5765
|
|
|
5766 my $cigar;
|
|
|
5767 if ($bowtie2){
|
|
|
5768 $cigar = $methylation_call_params->{$id}->{CIGAR}; # Actual CIGAR string reported by Bowtie 2
|
|
|
5769 }
|
|
|
5770 else{
|
|
|
5771 $cigar = length($actual_seq) . "M"; # Bowtie 1 output does not contain indels (only matches and mismatches)
|
|
|
5772 }
|
|
|
5773
|
|
|
5774 #####
|
|
|
5775
|
|
|
5776 my $rnext = "*"; # Paired-end variable
|
|
|
5777
|
|
|
5778 #####
|
|
|
5779
|
|
|
5780 my $pnext = 0; # Paired-end variable
|
|
|
5781
|
|
|
5782 #####
|
|
|
5783
|
|
|
5784 my $tlen = 0; # Paired-end variable
|
|
|
5785
|
|
|
5786 #####
|
|
|
5787
|
|
|
5788 if ($read_conversion eq 'CT'){
|
|
|
5789 $ref_seq = substr($ref_seq, 0, length($ref_seq) - 2); # Removes additional nucleotides from the 3' end. This only works for the original top or bottom strands
|
|
|
5790 }
|
|
|
5791 else{
|
|
|
5792 $ref_seq = substr($ref_seq, 2, length($ref_seq) - 2); # Removes additional nucleotides from the 5' end. This works for the complementary strands in non-directional libraries
|
|
|
5793 }
|
|
|
5794
|
|
|
5795 if ($strand eq '-'){
|
|
|
5796 $actual_seq = revcomp($actual_seq); # Sequence represented on the forward genomic strand
|
|
|
5797 $ref_seq = revcomp($ref_seq); # Required for comparison with actual sequence
|
|
|
5798 $qual = reverse $qual; # if the sequence was reverse-complemented the quality string needs to be reversed as well
|
|
|
5799 }
|
|
|
5800
|
|
|
5801 #####
|
|
|
5802
|
|
|
5803 my $hemming_dist = hemming_dist($actual_seq,$ref_seq); # Edit distance to the reference, i.e. minimal number of one-nucleotide edits needed to transform the read string
|
|
|
5804 # into the reference string. hemming_dist()
|
|
|
5805 if ($bowtie2){
|
|
|
5806 $hemming_dist += $methylation_call_params->{$id}->{indels}; # Adding the number of inserted/deleted bases which we parsed while getting the genomic sequence
|
|
|
5807 }
|
|
|
5808
|
|
|
5809 my $NM_tag = "NM:i:$hemming_dist"; # Optional tag NM: edit distance based on nucleotide differences
|
|
|
5810
|
|
|
5811 #####
|
|
|
5812
|
|
|
5813 my $XX_tag = make_mismatch_string($actual_seq, $ref_seq); # Optional tag XX: string providing mismatched reference bases in the alignment (NO indel information!)
|
|
|
5814
|
|
|
5815 #####
|
|
|
5816
|
|
|
5817 my $XM_tag; # Optional tag XM: Methylation Call String
|
|
|
5818 if ($strand eq '+'){
|
|
|
5819 $XM_tag = "XM:Z:$methcall";
|
|
|
5820 }
|
|
|
5821 elsif ($strand eq '-'){
|
|
|
5822 $XM_tag = 'XM:Z:'.reverse $methcall; # if the sequence was reverse-complemented the methylation call string needs to be reversed as well
|
|
|
5823 }
|
|
|
5824
|
|
|
5825 #####
|
|
|
5826
|
|
|
5827 my $XR_tag = "XR:Z:$read_conversion"; # Optional tag XR: Read Conversion
|
|
|
5828
|
|
|
5829 #####
|
|
|
5830
|
|
|
5831 my $XG_tag = "XG:Z:$genome_conversion"; # Optional tag XG: Genome Conversion
|
|
|
5832
|
|
|
5833 #####
|
|
|
5834
|
|
|
5835 # SAM format: QNAME, FLAG, RNAME, 1-based POS, MAPQ, CIGAR, RNEXT, PNEXT, TLEN, SEQ, QUAL, optional fields
|
|
|
5836 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";
|
|
|
5837 }
|
|
|
5838
|
|
|
5839
|
|
|
5840 sub paired_end_SAM_output{
|
|
|
5841 my ($id,$actual_seq_1,$actual_seq_2,$methylation_call_params,$qual_1,$qual_2) = @_;
|
|
|
5842 my $strand_1 = $methylation_call_params->{$id}->{alignment_read_1}; # Bowtie 1 only reports the read 1 alignment strand
|
|
|
5843 my $strand_2 = $methylation_call_params->{$id}->{alignment_read_2};
|
|
|
5844 my $chr = $methylation_call_params->{$id}->{chromosome};
|
|
|
5845 my $ref_seq_1 = $methylation_call_params->{$id}->{unmodified_genomic_sequence_1};
|
|
|
5846 my $ref_seq_2 = $methylation_call_params->{$id}->{unmodified_genomic_sequence_2};
|
|
|
5847 my $methcall_1 = $methylation_call_params->{$id}->{methylation_call_1};
|
|
|
5848 my $methcall_2 = $methylation_call_params->{$id}->{methylation_call_2};
|
|
|
5849 my $read_conversion_1 = $methylation_call_params->{$id}->{read_conversion_1};
|
|
|
5850 my $read_conversion_2 = $methylation_call_params->{$id}->{read_conversion_2};
|
|
|
5851 my $genome_conversion = $methylation_call_params->{$id}->{genome_conversion};
|
|
|
5852 my $number_of_mismatches_1 = $methylation_call_params->{$id}->{number_of_mismatches_1}; # only needed for custom allele-specific output, not the default!
|
|
|
5853 my $number_of_mismatches_2 = $methylation_call_params->{$id}->{number_of_mismatches_2};
|
|
|
5854
|
|
|
5855 my $id_1 = $id.'/1';
|
|
|
5856 my $id_2 = $id.'/2';
|
|
|
5857
|
|
|
5858 # Allows all degenerate nucleotide sequences in reference genome
|
|
|
5859 die "Reference sequence ($ref_seq_1) contains invalid nucleotides!\n" if $ref_seq_1 =~ /[^ACTGNRYMKSWBDHV]/i;
|
|
|
5860 die "Reference sequence ($ref_seq_2) contains invalid nucleotides!\n" if $ref_seq_2 =~ /[^ACTGNRYMKSWBDHV]/i;
|
|
|
5861
|
|
|
5862 my $index; # used to store the srand origin of the alignment in a less convoluted way
|
|
|
5863
|
|
|
5864 if ($read_conversion_1 eq 'CT' and $genome_conversion eq 'CT'){
|
|
|
5865 $index = 0; ## this is OT (original top strand)
|
|
|
5866 }
|
|
|
5867 elsif ($read_conversion_1 eq 'GA' and $genome_conversion eq 'GA'){
|
|
|
5868 $index = 1; ## this is CTOB (complementary to OB)
|
|
|
5869 }
|
|
|
5870 elsif ($read_conversion_1 eq 'GA' and $genome_conversion eq 'CT'){
|
|
|
5871 $index = 2; ## this is CTOT (complementary to OT)
|
|
|
5872 }
|
|
|
5873 elsif ($read_conversion_1 eq 'CT' and $genome_conversion eq 'GA'){
|
|
|
5874 $index = 3; ## this is OB (original bottom)
|
|
|
5875 }
|
|
|
5876 else {
|
|
|
5877 die "Unexpected combination of read 1 and genome conversion: $read_conversion_1 / $genome_conversion\n";
|
|
|
5878 }
|
|
|
5879
|
|
|
5880 ### we need to remove 2 bp of the genomic sequence as we were extracting read + 2bp long fragments to make a methylation call at the
|
|
|
5881 ### first or last position.
|
|
|
5882
|
|
|
5883 if ($index == 0 or $index == 3){ # OT or OB
|
|
|
5884 $ref_seq_1 = substr($ref_seq_1,0,length($ref_seq_1)-2);
|
|
|
5885 $ref_seq_2 = substr($ref_seq_2,2,length($ref_seq_2)-2);
|
|
|
5886 }
|
|
|
5887 else{ # CTOT or CTOB
|
|
|
5888 $ref_seq_1 = substr($ref_seq_1,2,length($ref_seq_1)-2);
|
|
|
5889 $ref_seq_2 = substr($ref_seq_2,0,length($ref_seq_2)-2);
|
|
|
5890 }
|
|
|
5891
|
|
|
5892 #####
|
|
|
5893
|
|
|
5894 my $start_read_1;
|
|
|
5895 my $start_read_2;
|
|
|
5896 # adjusting end positions
|
|
|
5897
|
|
|
5898 if ($bowtie2){
|
|
|
5899 $start_read_1 = $methylation_call_params->{$id}->{position_1};
|
|
|
5900 $start_read_2 = $methylation_call_params->{$id}->{position_2};
|
|
|
5901 }
|
|
|
5902 else{ # Bowtie 1 output. $strand_1 stores the alignment of Read 1
|
|
|
5903 if ($strand_1 eq '+'){ # Read 1 aligns to the + strand
|
|
|
5904 $start_read_1 = $methylation_call_params->{$id}->{start_seq_1};
|
|
|
5905 $start_read_2 = $methylation_call_params->{$id}->{alignment_end} - length ($actual_seq_2) + 1;
|
|
|
5906 }
|
|
|
5907 else{ # read 1 is on the - strand
|
|
|
5908 $start_read_1 = $methylation_call_params->{$id}->{alignment_end} - length ($actual_seq_1) + 1;
|
|
|
5909 $start_read_2 = $methylation_call_params->{$id}->{start_seq_1};
|
|
|
5910 }
|
|
|
5911 }
|
|
|
5912
|
|
|
5913 #####
|
|
|
5914
|
|
|
5915 my $end_read_1;
|
|
|
5916 my $end_read_2;
|
|
|
5917 # adjusting end positions
|
|
|
5918
|
|
|
5919 if ($bowtie2){
|
|
|
5920 $end_read_1 = $methylation_call_params->{$id}->{end_position_1};
|
|
|
5921 $end_read_2 = $methylation_call_params->{$id}->{end_position_2};
|
|
|
5922 }
|
|
|
5923 else{ # Bowtie 1 output. $strand_1 stores the alignment of Read 1
|
|
|
5924 if ($strand_1 eq '+'){ # Read 1 aligns to the + strand
|
|
|
5925 $end_read_1 = $methylation_call_params->{$id}->{start_seq_1} + length ($actual_seq_1)-1;
|
|
|
5926 $end_read_2 = $methylation_call_params->{$id}->{alignment_end};
|
|
|
5927 }
|
|
|
5928 else{
|
|
|
5929 $end_read_1 = $methylation_call_params->{$id}->{alignment_end};
|
|
|
5930 $end_read_2 = $methylation_call_params->{$id}->{start_seq_1} + length ($actual_seq_2)-1;
|
|
|
5931 }
|
|
|
5932 }
|
|
|
5933
|
|
|
5934 #####
|
|
|
5935
|
|
|
5936 ### 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"
|
|
|
5937 ## FLAG: bitwise FLAG. Each bit is explained in the following table:
|
|
|
5938 ## Bit Description Comment Value
|
|
|
5939 ## 0x1 template having multiple segments in sequencing 0: single-end 1: paired end value: 2^^0 ( 1)
|
|
|
5940 ## 0x2 each segment properly aligned according to the aligner true only for paired-end alignments value: 2^^1 ( 2)
|
|
|
5941 ## 0x4 segment unmapped --- ---
|
|
|
5942 ## 0x8 next segment in the template unmapped --- ---
|
|
|
5943 ## 0x10 SEQ being reverse complemented - strand alignment value: 2^^4 ( 16)
|
|
|
5944 ## 0x20 SEQ of the next segment in the template being reversed + strand alignment value: 2^^5 ( 32)
|
|
|
5945 ## 0x40 the first segment in the template read 1 value: 2^^6 ( 64)
|
|
|
5946 ## 0x80 the last segment in the template read 2 value: 2^^7 (128)
|
|
|
5947 ## 0x100 secondary alignment --- ---
|
|
|
5948 ## 0x200 not passing quality controls --- ---
|
|
|
5949 ## 0x400 PCR or optical duplicate --- ---
|
|
|
5950
|
|
|
5951 ### 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
|
|
|
5952
|
|
|
5953 # strands OT and CTOT will be treated as aligning to the top strand (both sequences are scored as aligning to the top strand)
|
|
|
5954 # strands OB and CTOB will be treated as aligning to the bottom strand (both sequences are scored as reverse complemented sequences)
|
|
|
5955
|
|
|
5956 my $flag_1; # FLAG variable used for SAM format
|
|
|
5957 my $flag_2;
|
|
|
5958
|
|
|
5959 if ($index == 0){ # OT
|
|
|
5960 $flag_1 = 67; # Read 1 is on the + strand (1+2+64) (Read 2 is technically reverse-complemented, but we do not score it)
|
|
|
5961 $flag_2 = 131; # Read 2 is on - strand but informative for the OT (1+2+128)
|
|
|
5962 }
|
|
|
5963 elsif ($index == 1){ # CTOB
|
|
|
5964 $flag_1 = 115; # Read 1 is on the + strand, we score for OB (1+2+16+32+64)
|
|
|
5965 $flag_2 = 179; # Read 2 is on the - strand (1+2+16+32+128)
|
|
|
5966 }
|
|
|
5967 elsif ($index == 2){ # CTOT
|
|
|
5968 $flag_1 = 67; # Read 1 is on the - strand (CTOT) strand, but we score it for OT (1+2+64)
|
|
|
5969 $flag_2 = 131; # Read 2 is on the + strand, score it for OT (1+2+128)
|
|
|
5970 }
|
|
|
5971 elsif ($index == 3){ # OB
|
|
|
5972 $flag_1 = 115; # Read 1 is on the - strand, we score for OB (1+2+16+32+64)
|
|
|
5973 $flag_2 = 179; # Read 2 is on the + strand (1+2+16+32+128)
|
|
|
5974 }
|
|
|
5975
|
|
|
5976 #####
|
|
|
5977
|
|
|
5978 my $mapq = 255; # Mapping quality is unavailable
|
|
|
5979
|
|
|
5980 #####
|
|
|
5981
|
|
|
5982 my $cigar_1;
|
|
|
5983 my $cigar_2;
|
|
|
5984
|
|
|
5985 if ($bowtie2){
|
|
|
5986 $cigar_1 = $methylation_call_params->{$id}->{CIGAR_1}; # Actual CIGAR string reported by Bowtie 2
|
|
|
5987 $cigar_2 = $methylation_call_params->{$id}->{CIGAR_2};
|
|
|
5988 }
|
|
|
5989 else{
|
|
|
5990 $cigar_1 = length($actual_seq_1) . "M"; # Assume no indels for Bowtie 1 mapping (only matches and mismatches)
|
|
|
5991 $cigar_2 = length($actual_seq_2) . "M";
|
|
|
5992 }
|
|
|
5993
|
|
|
5994 #####
|
|
|
5995
|
|
|
5996 my $rnext = '='; # Chromosome of mate; applies to both reads
|
|
|
5997
|
|
|
5998 #####
|
|
|
5999
|
|
|
6000 my $pnext_1 = $start_read_2; # Leftmost position of mate
|
|
|
6001 my $pnext_2 = $start_read_1;
|
|
|
6002
|
|
|
6003 #####
|
|
|
6004
|
|
|
6005 my $tlen_1; # signed observed Template LENgth (or inferred fragment size)
|
|
|
6006 my $tlen_2;
|
|
|
6007
|
|
|
6008 if ($bowtie2){
|
|
|
6009
|
|
|
6010 if ($start_read_1 <= $start_read_2){
|
|
|
6011
|
|
|
6012 # Read 1 alignment is leftmost
|
|
|
6013
|
|
|
6014 if ($end_read_2 >= $end_read_1){
|
|
|
6015
|
|
|
6016 # -------------------------> read 1 reads overlapping
|
|
|
6017 # <------------------------- read 2
|
|
|
6018 #
|
|
|
6019 # or
|
|
|
6020 #
|
|
|
6021 # -------------------------> read 1
|
|
|
6022 # <----------------------- read 2 read 2 contained within read 1
|
|
|
6023 #
|
|
|
6024 # or
|
|
|
6025 #
|
|
|
6026 # -------------------------> read 1 reads 1 and 2 exactly overlapping
|
|
|
6027 # <------------------------- read 2
|
|
|
6028 #
|
|
|
6029
|
|
|
6030 # dovetailing of reads is not enabled for Bowtie 2 alignments
|
|
|
6031
|
|
|
6032 $tlen_1 = $end_read_2 - $start_read_1 + 1; # Leftmost read has a + sign,
|
|
|
6033 $tlen_2 = $start_read_1 - $end_read_2 - 1; # Rightmost read has a - sign
|
|
|
6034 }
|
|
|
6035 elsif ($end_read_2 < $end_read_1){
|
|
|
6036
|
|
|
6037 # -------------------------> read 1
|
|
|
6038 # <----------- read 2 read 2 contained within read 1
|
|
|
6039 #
|
|
|
6040 # or
|
|
|
6041 #
|
|
|
6042 # -------------------------> read 1
|
|
|
6043 # <----------- read 2 read 2 contained within read 1
|
|
|
6044
|
|
|
6045 # start and end of read 2 are fully contained within read 1
|
|
|
6046 $tlen_1 = 0; # Set as 0 when the information is unavailable
|
|
|
6047 $tlen_2 = 0; # Set as 0 when the information is unavailable
|
|
|
6048 }
|
|
|
6049
|
|
|
6050 }
|
|
|
6051
|
|
|
6052 elsif ($start_read_2 < $start_read_1){
|
|
|
6053
|
|
|
6054 if ($end_read_1 >= $end_read_2){
|
|
|
6055
|
|
|
6056 # Read 2 alignment is leftmost
|
|
|
6057
|
|
|
6058 # -------------------------> read 2 reads overlapping
|
|
|
6059 # <------------------------- read 1
|
|
|
6060 #
|
|
|
6061 # or
|
|
|
6062 #
|
|
|
6063 # -------------------------> read 2
|
|
|
6064 # <----------------------- read 1 read 1 contained within read 2
|
|
|
6065 #
|
|
|
6066 #
|
|
|
6067
|
|
|
6068 $tlen_2 = $end_read_1 - $start_read_2 + 1; # Leftmost read has a + sign,
|
|
|
6069 $tlen_1 = $start_read_2 - $end_read_1 - 1; # Rightmost read has a - sign
|
|
|
6070 }
|
|
|
6071 elsif ($end_read_1 < $end_read_2){
|
|
|
6072
|
|
|
6073 # -------------------------> read 2
|
|
|
6074 # <----------- read 1 read 1 contained within read 2
|
|
|
6075 #
|
|
|
6076 # or
|
|
|
6077 #
|
|
|
6078 # -------------------------> read 2
|
|
|
6079 # <----------- read 1 read 1 contained within read 2
|
|
|
6080
|
|
|
6081 # start and end of read 1 are fully contained within read 2
|
|
|
6082 $tlen_1 = 0; # Set as 0 when the information is unavailable
|
|
|
6083 $tlen_2 = 0; # Set as 0 when the information is unavailable
|
|
|
6084 }
|
|
|
6085 }
|
|
|
6086 }
|
|
|
6087
|
|
|
6088 else{ # Bowtie 1
|
|
|
6089
|
|
|
6090 if ($end_read_2 >= $end_read_1){
|
|
|
6091 # Read 1 alignment is leftmost
|
|
|
6092 # -------------------------> read 1
|
|
|
6093 # <------------------------- read 2
|
|
|
6094 # this is the most extreme case for Bowtie 1 alignments, reads do not contain each other, also no dovetailing
|
|
|
6095
|
|
|
6096 $tlen_1 = $end_read_2 - $start_read_1 + 1; # Leftmost read has a + sign,
|
|
|
6097 $tlen_2 = $start_read_1 - $end_read_2 - 1; # Rightmost read has a - sign
|
|
|
6098 }
|
|
|
6099 else{
|
|
|
6100 # Read 2 alignment is leftmost
|
|
|
6101 # -------------------------> read 2
|
|
|
6102 # <------------------------- read 1
|
|
|
6103 # this is the most extreme case for Bowtie 1 alignments, reads do not contain each other, also no dovetailing
|
|
|
6104
|
|
|
6105 $tlen_2 = $end_read_1 - $start_read_2 + 1; # Leftmost read has a + sign,
|
|
|
6106 $tlen_1 = $start_read_2 - $end_read_1 - 1; # Rightmost read has a - sign
|
|
|
6107 }
|
|
|
6108 }
|
|
|
6109
|
|
|
6110 #####
|
|
|
6111
|
|
|
6112 # adjusting the strand of the sequence before we use them to generate mismatch strings
|
|
|
6113 if ($strand_1 eq '-'){
|
|
|
6114 $actual_seq_1 = revcomp($actual_seq_1); # Sequence represented on the forward genomic strand
|
|
|
6115 $ref_seq_1 = revcomp($ref_seq_1); # Required for comparison with actual sequence
|
|
|
6116 $qual_1 = reverse $qual_1; # we need to reverse the quality string as well
|
|
|
6117 }
|
|
|
6118 if ($strand_2 eq '-'){
|
|
|
6119 $actual_seq_2 = revcomp($actual_seq_2); # Mate sequence represented on the forward genomic strand
|
|
|
6120 $ref_seq_2 = revcomp($ref_seq_2); # Required for comparison with actual sequence
|
|
|
6121 $qual_2 = reverse $qual_2; # If the sequence gets reverse complemented we reverse the quality string as well
|
|
|
6122 }
|
|
|
6123
|
|
|
6124 # print "$actual_seq_1\n$ref_seq_1\n\n";
|
|
|
6125 # print "$actual_seq_2\n$ref_seq_2\n\n";
|
|
|
6126
|
|
|
6127 #####
|
|
|
6128
|
|
|
6129 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
|
|
|
6130 my $hemming_dist_2 = hemming_dist($actual_seq_2,$ref_seq_2);
|
|
|
6131 if ($bowtie2){
|
|
|
6132 $hemming_dist_1 += $methylation_call_params->{$id}->{indels_1}; # Adding the number of inserted/deleted bases which we parsed while getting the genomic sequence
|
|
|
6133 $hemming_dist_2 += $methylation_call_params->{$id}->{indels_2}; # Adding the number of inserted/deleted bases which we parsed while getting the genomic sequence
|
|
|
6134 }
|
|
|
6135 my $NM_tag_1 = "NM:i:$hemming_dist_1"; # Optional tag NM: edit distance based on nucleotide differences
|
|
|
6136 my $NM_tag_2 = "NM:i:$hemming_dist_2"; # Optional tag NM: edit distance based on nucleotide differences
|
|
|
6137
|
|
|
6138 #####
|
|
|
6139
|
|
|
6140 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!)
|
|
|
6141 my $XX_tag_2 = make_mismatch_string($actual_seq_2,$ref_seq_2);
|
|
|
6142
|
|
|
6143 #####
|
|
|
6144
|
|
|
6145 my $XM_tag_1; # Optional tag XM: Methylation call string
|
|
|
6146 my $XM_tag_2;
|
|
|
6147
|
|
|
6148 if ($strand_1 eq '-'){
|
|
|
6149 $XM_tag_1 = 'XM:Z:'.reverse $methcall_1; # Needs to be reversed if the sequence was reverse complemented
|
|
|
6150 }
|
|
|
6151 else{
|
|
|
6152 $XM_tag_1 = "XM:Z:$methcall_1";
|
|
|
6153 }
|
|
|
6154
|
|
|
6155 if ($strand_2 eq '-'){
|
|
|
6156 $XM_tag_2 = 'XM:Z:'.reverse $methcall_2; # Needs to be reversed if the sequence was reverse complemented
|
|
|
6157 }
|
|
|
6158 else{
|
|
|
6159 $XM_tag_2 = "XM:Z:$methcall_2";
|
|
|
6160 }
|
|
|
6161
|
|
|
6162 #####
|
|
|
6163
|
|
|
6164 my $XR_tag_1 = "XR:Z:$read_conversion_1"; # Optional tag XR: Read 1 conversion state
|
|
|
6165 my $XR_tag_2 = "XR:Z:$read_conversion_2"; # Optional tag XR: Read 2 conversion state
|
|
|
6166
|
|
|
6167 #####
|
|
|
6168
|
|
|
6169 my $XG_tag = "XG:Z:$genome_conversion"; # Optional tag XG: Genome Conversion state; valid for both reads
|
|
|
6170
|
|
|
6171 #####
|
|
|
6172
|
|
|
6173 # SAM format: QNAME, FLAG, RNAME, 1-based POS, MAPQ, CIGAR, RNEXT, PNEXT, TLEN, SEQ, QUAL, optional fields
|
|
|
6174 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,$XG_tag)), "\n";
|
|
|
6175 print OUT join("\t", ($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";
|
|
|
6176 }
|
|
|
6177
|
|
|
6178 sub revcomp{
|
|
|
6179 my $seq = shift or die "Missing seq to reverse complement\n";
|
|
|
6180 $seq = reverse $seq;
|
|
|
6181 $seq =~ tr/ACTGactg/TGACTGAC/;
|
|
|
6182 return $seq;
|
|
|
6183 }
|
|
|
6184
|
|
|
6185 sub hemming_dist{
|
|
|
6186 my $matches = 0;
|
|
|
6187 my @actual_seq = split //,(shift @_);
|
|
|
6188 my @ref_seq = split //,(shift @_);
|
|
|
6189 foreach (0..$#actual_seq){
|
|
|
6190 ++$matches if ($actual_seq[$_] eq $ref_seq[$_]);
|
|
|
6191 }
|
|
|
6192 return my $hd = scalar @actual_seq - $matches;
|
|
|
6193 }
|
|
|
6194
|
|
|
6195 sub make_mismatch_string{
|
|
|
6196 my $actual_seq = shift or die "Missing actual sequence";
|
|
|
6197 my $ref_seq = shift or die "Missing reference sequence";
|
|
|
6198 my $XX_tag = "XX:Z:";
|
|
|
6199 my $tmp = ($actual_seq ^ $ref_seq); # Bitwise comparison
|
|
|
6200 my $prev_mm_pos = 0;
|
|
|
6201 while($tmp =~ /[^\0]/g){ # Where bitwise comparison showed a difference
|
|
|
6202 my $nuc_match = pos($tmp) - $prev_mm_pos - 1; # Generate number of nucleotide that matches since last mismatch
|
|
|
6203 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
|
|
|
6204 $XX_tag .= "$nuc_match" if $nuc_match > 0; # Ignore if mismatches are adjacent to each other
|
|
|
6205 $XX_tag .= "$nuc_mm" if defined $nuc_mm; # Ignore if there is no mismatch (prevents uninitialized string concatenation)
|
|
|
6206 $prev_mm_pos = pos($tmp); # Position of last mismatch
|
|
|
6207 }
|
|
|
6208 my $end_matches = length($ref_seq) - $prev_mm_pos; # Provides number of matches from last mismatch till end of sequence
|
|
|
6209 $XX_tag .= "$end_matches" if $end_matches > 0; # Ignore if mismatch is at the end of sequence
|
|
|
6210 return $XX_tag;
|
|
|
6211 }
|
|
|
6212
|
|
|
6213
|
|
|
6214
|
|
|
6215 sub print_helpfile{
|
|
|
6216 print << "HOW_TO";
|
|
|
6217
|
|
|
6218
|
|
|
6219 This program is free software: you can redistribute it and/or modify
|
|
|
6220 it under the terms of the GNU General Public License as published by
|
|
|
6221 the Free Software Foundation, either version 3 of the License, or
|
|
|
6222 (at your option) any later version.
|
|
|
6223
|
|
|
6224 This program is distributed in the hope that it will be useful,
|
|
|
6225 but WITHOUT ANY WARRANTY; without even the implied warranty of
|
|
|
6226 MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the
|
|
|
6227 GNU General Public License for more details.
|
|
|
6228 You should have received a copy of the GNU General Public License
|
|
|
6229 along with this program. If not, see <http://www.gnu.org/licenses/>.
|
|
|
6230
|
|
|
6231
|
|
|
6232
|
|
|
6233 DESCRIPTION
|
|
|
6234
|
|
|
6235
|
|
|
6236 The following is a brief description of command line options and arguments to control the Bismark
|
|
|
6237 bisulfite mapper and methylation caller. Bismark takes in FastA or FastQ files and aligns the
|
|
|
6238 reads to a specified bisulfite genome. Sequence reads are transformed into a bisulfite converted forward strand
|
|
|
6239 version (C->T conversion) or into a bisulfite treated reverse strand (G->A conversion of the forward strand).
|
|
|
6240 Each of these reads are then aligned to bisulfite treated forward strand index of a reference genome
|
|
|
6241 (C->T converted) and a bisulfite treated reverse strand index of the genome (G->A conversion of the
|
|
|
6242 forward strand, by doing this alignments will produce the same positions). These 4 instances of Bowtie (1 or 2)
|
|
|
6243 are run in parallel. The sequence file(s) are then read in again sequence by sequence to pull out the original
|
|
|
6244 sequence from the genome and determine if there were any protected C's present or not.
|
|
|
6245
|
|
|
6246 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
|
|
|
6247 re-enabled by using --non_directional.
|
|
|
6248
|
|
|
6249 The final output of Bismark is in SAM format by default. For Bowtie 1 one can alos choose to report the old
|
|
|
6250 'vanilla' output format, which is a single tab delimited file with all sequences that have a unique best
|
|
|
6251 alignment to any of the 4 possible strands of a bisulfite PCR product. Both formats are described in more detail below.
|
|
|
6252
|
|
|
6253
|
|
|
6254 USAGE: bismark [options] <genome_folder> {-1 <mates1> -2 <mates2> | <singles>}
|
|
|
6255
|
|
|
6256
|
|
|
6257 ARGUMENTS:
|
|
|
6258
|
|
|
6259 <genome_folder> The path to the folder containing the unmodified reference genome
|
|
|
6260 as well as the subfolders created by the Bismark_Genome_Preparation
|
|
|
6261 script (/Bisulfite_Genome/CT_conversion/ and /Bisulfite_Genome/GA_conversion/).
|
|
|
6262 Bismark expects one or more fastA files in this folder (file extension: .fa
|
|
|
6263 or .fasta). The path can be relative or absolute.
|
|
|
6264
|
|
|
6265 -1 <mates1> Comma-separated list of files containing the #1 mates (filename usually includes
|
|
|
6266 "_1"), e.g. flyA_1.fq,flyB_1.fq). Sequences specified with this option must
|
|
|
6267 correspond file-for-file and read-for-read with those specified in <mates2>.
|
|
|
6268 Reads may be a mix of different lengths. Bismark will produce one mapping result
|
|
|
6269 and one report file per paired-end input file pair.
|
|
|
6270
|
|
|
6271 -2 <mates2> Comma-separated list of files containing the #2 mates (filename usually includes
|
|
|
6272 "_2"), e.g. flyA_1.fq,flyB_1.fq). Sequences specified with this option must
|
|
|
6273 correspond file-for-file and read-for-read with those specified in <mates1>.
|
|
|
6274 Reads may be a mix of different lengths.
|
|
|
6275
|
|
|
6276 <singles> A comma- or space-separated list of files containing the reads to be aligned (e.g.
|
|
|
6277 lane1.fq,lane2.fq lane3.fq). Reads may be a mix of different lengths. Bismark will
|
|
|
6278 produce one mapping result and one report file per input file.
|
|
|
6279
|
|
|
6280
|
|
|
6281 OPTIONS:
|
|
|
6282
|
|
|
6283
|
|
|
6284 Input:
|
|
|
6285
|
|
|
6286 -q/--fastq The query input files (specified as <mate1>,<mate2> or <singles> are FASTQ
|
|
|
6287 files (usually having extension .fg or .fastq). This is the default. See also
|
|
|
6288 --solexa-quals.
|
|
|
6289
|
|
|
6290 -f/--fasta The query input files (specified as <mate1>,<mate2> or <singles> are FASTA
|
|
|
6291 files (usually havin extension .fa, .mfa, .fna or similar). All quality values
|
|
|
6292 are assumed to be 40 on the Phred scale.
|
|
|
6293
|
|
|
6294 -s/--skip <int> Skip (i.e. do not align) the first <int> reads or read pairs from the input.
|
|
|
6295
|
|
|
6296 -u/--upto <int> Only aligns the first <int> reads or read pairs from the input. Default: no limit.
|
|
|
6297
|
|
|
6298 --phred33-quals FASTQ qualities are ASCII chars equal to the Phred quality plus 33. Default: on.
|
|
|
6299
|
|
|
6300 --phred64-quals FASTQ qualities are ASCII chars equal to the Phred quality plus 64. Default: off.
|
|
|
6301
|
|
|
6302 --solexa-quals Convert FASTQ qualities from solexa-scaled (which can be negative) to phred-scaled
|
|
|
6303 (which can't). The formula for conversion is:
|
|
|
6304 phred-qual = 10 * log(1 + 10 ** (solexa-qual/10.0)) / log(10). Used with -q. This
|
|
|
6305 is usually the right option for use with (unconverted) reads emitted by the GA
|
|
|
6306 Pipeline versions prior to 1.3. Works only for Bowtie 1. Default: off.
|
|
|
6307
|
|
|
6308 --solexa1.3-quals Same as --phred64-quals. This is usually the right option for use with (unconverted)
|
|
|
6309 reads emitted by GA Pipeline version 1.3 or later. Default: off.
|
|
|
6310
|
|
|
6311 --path_to_bowtie The full path </../../> to the Bowtie (1 or 2) installation on your system. If not
|
|
|
6312 specified it is assumed that Bowtie (1 or 2) is in the PATH.
|
|
|
6313
|
|
|
6314
|
|
|
6315 Alignment:
|
|
|
6316
|
|
|
6317 -n/--seedmms <int> The maximum number of mismatches permitted in the "seed", i.e. the first L base pairs
|
|
|
6318 of the read (where L is set with -l/--seedlen). This may be 0, 1, 2 or 3 and the
|
|
|
6319 default is 1. This option is only available for Bowtie 1 (for Bowtie 2 see -N).
|
|
|
6320
|
|
|
6321 -l/--seedlen The "seed length"; i.e., the number of bases of the high quality end of the read to
|
|
|
6322 which the -n ceiling applies. The default is 28. Bowtie (and thus Bismark) is faster for
|
|
|
6323 larger values of -l. This option is only available for Bowtie 1 (for Bowtie 2 see -L).
|
|
|
6324
|
|
|
6325 -e/--maqerr <int> Maximum permitted total of quality values at all mismatched read positions throughout
|
|
|
6326 the entire alignment, not just in the "seed". The default is 70. Like Maq, bowtie rounds
|
|
|
6327 quality values to the nearest 10 and saturates at 30. This value is not relevant for
|
|
|
6328 Bowtie 2.
|
|
|
6329
|
|
|
6330 --chunkmbs <int> The number of megabytes of memory a given thread is given to store path descriptors in
|
|
|
6331 --best mode. Best-first search must keep track of many paths at once to ensure it is
|
|
|
6332 always extending the path with the lowest cumulative cost. Bowtie tries to minimize the
|
|
|
6333 memory impact of the descriptors, but they can still grow very large in some cases. If
|
|
|
6334 you receive an error message saying that chunk memory has been exhausted in --best mode,
|
|
|
6335 try adjusting this parameter up to dedicate more memory to the descriptors. This value
|
|
|
6336 is not relevant for Bowtie 2. Default: 512.
|
|
|
6337
|
|
|
6338 -I/--minins <int> The minimum insert size for valid paired-end alignments. E.g. if -I 60 is specified and
|
|
|
6339 a paired-end alignment consists of two 20-bp alignments in the appropriate orientation
|
|
|
6340 with a 20-bp gap between them, that alignment is considered valid (as long as -X is also
|
|
|
6341 satisfied). A 19-bp gap would not be valid in that case. Default: 0.
|
|
|
6342
|
|
|
6343 -X/--maxins <int> The maximum insert size for valid paired-end alignments. E.g. if -X 100 is specified and
|
|
|
6344 a paired-end alignment consists of two 20-bp alignments in the proper orientation with a
|
|
|
6345 60-bp gap between them, that alignment is considered valid (as long as -I is also satisfied).
|
|
|
6346 A 61-bp gap would not be valid in that case. Default: 500.
|
|
|
6347
|
|
|
6348
|
|
|
6349 Bowtie 1 Reporting:
|
|
|
6350
|
|
|
6351 -k <2> Due to the way Bismark works Bowtie will report up to 2 valid alignments. This option
|
|
|
6352 will be used by default.
|
|
|
6353
|
|
|
6354 --best Make Bowtie guarantee that reported singleton alignments are "best" in terms of stratum
|
|
|
6355 (i.e. number of mismatches, or mismatches in the seed in the case if -n mode) and in
|
|
|
6356 terms of the quality; e.g. a 1-mismatch alignment where the mismatch position has Phred
|
|
|
6357 quality 40 is preferred over a 2-mismatch alignment where the mismatched positions both
|
|
|
6358 have Phred quality 10. When --best is not specified, Bowtie may report alignments that
|
|
|
6359 are sub-optimal in terms of stratum and/or quality (though an effort is made to report
|
|
|
6360 the best alignment). --best mode also removes all strand bias. Note that --best does not
|
|
|
6361 affect which alignments are considered "valid" by Bowtie, only which valid alignments
|
|
|
6362 are reported by Bowtie. Bowtie is about 1-2.5 times slower when --best is specified.
|
|
|
6363 Default: on.
|
|
|
6364
|
|
|
6365 --no_best Disables the --best option which is on by default. This can speed up the alignment process,
|
|
|
6366 e.g. for testing purposes, but for credible results it is not recommended to disable --best.
|
|
|
6367
|
|
|
6368
|
|
|
6369 Output:
|
|
|
6370
|
|
|
6371 --non_directional The sequencing library was constructed in a non strand-specific manner, alignments to all four
|
|
|
6372 bisulfite strands will be reported. Default: OFF.
|
|
|
6373
|
|
|
6374 (The current Illumina protocol for BS-Seq is directional, in which case the strands complementary
|
|
|
6375 to the original strands are merely theoretical and should not exist in reality. Specifying directional
|
|
|
6376 alignments (which is the default) will only run 2 alignment threads to the original top (OT)
|
|
|
6377 or bottom (OB) strands in parallel and report these alignments. This is the recommended option
|
|
|
6378 for sprand-specific libraries).
|
|
|
6379
|
|
|
6380 --sam-no-hd Suppress SAM header lines (starting with @). This might be useful when very large input files are
|
|
|
6381 split up into several smaller files to run concurrently and the output files are to be merged.
|
|
|
6382
|
|
|
6383 --quiet Print nothing besides alignments.
|
|
|
6384
|
|
|
6385 --vanilla Performs bisulfite mapping with Bowtie 1 and prints the 'old' output (as in Bismark 0.5.X) instead
|
|
|
6386 of SAM format output.
|
|
|
6387
|
|
|
6388 -un/--unmapped Write all reads that could not be aligned to a file in the output directory. Written reads will
|
|
|
6389 appear as they did in the input, without any translation of quality values that may have
|
|
|
6390 taken place within Bowtie or Bismark. Paired-end reads will be written to two parallel files with _1
|
|
|
6391 and _2 inserted in their filenames, i.e. _unmapped_reads_1.txt and unmapped_reads_2.txt. Reads
|
|
|
6392 with more than one valid alignment with the same number of lowest mismatches (ambiguous mapping)
|
|
|
6393 are also written to _unmapped_reads.txt unless the option --ambiguous is specified as well.
|
|
|
6394
|
|
|
6395 --ambiguous Write all reads which produce more than one valid alignment with the same number of lowest
|
|
|
6396 mismatches or other reads that fail to align uniquely to a file in the output directory.
|
|
|
6397 Written reads will appear as they did in the input, without any of the translation of quality
|
|
|
6398 values that may have taken place within Bowtie or Bismark. Paired-end reads will be written to two
|
|
|
6399 parallel files with _1 and _2 inserted in theit filenames, i.e. _ambiguous_reads_1.txt and
|
|
|
6400 _ambiguous_reads_2.txt. These reads are not written to the file specified with --un.
|
|
|
6401
|
|
|
6402 -o/--output_dir <dir> Write all output files into this directory. By default the output files will be written into
|
|
|
6403 the same folder as the input file(s). If the specified folder does not exist, Bismark will attempt
|
|
|
6404 to create it first. The path to the output folder can be either relative or absolute.
|
|
|
6405
|
|
|
6406 --temp_dir <dir> Write temporary files to this directory instead of into the same directory as the input files. If
|
|
|
6407 the specified folder does not exist, Bismark will attempt to create it first. The path to the
|
|
|
6408 temporary folder can be either relative or absolute.
|
|
|
6409
|
|
|
6410
|
|
|
6411
|
|
|
6412 Other:
|
|
|
6413
|
|
|
6414 -h/--help Displays this help file.
|
|
|
6415
|
|
|
6416 -v/--version Displays version information.
|
|
|
6417
|
|
|
6418
|
|
|
6419 BOWTIE 2 SPECIFIC OPTIONS
|
|
|
6420
|
|
|
6421 --bowtie2 Uses Bowtie 2 instead of Bowtie 1. Bismark limits Bowtie 2 to only perform end-to-end
|
|
|
6422 alignments, i.e. searches for alignments involving all read characters (also called
|
|
|
6423 untrimmed or unclipped alignments). Bismark assumes that raw sequence data is adapter
|
|
|
6424 and/or quality trimmed where appropriate. Default: off.
|
|
|
6425
|
|
|
6426 Bowtie 2 alignment options:
|
|
|
6427
|
|
|
6428 -N <int> Sets the number of mismatches to allowed in a seed alignment during multiseed alignment.
|
|
|
6429 Can be set to 0 or 1. Setting this higher makes alignment slower (often much slower)
|
|
|
6430 but increases sensitivity. Default: 0. This option is only available for Bowtie 2 (for
|
|
|
6431 Bowtie 1 see -n).
|
|
|
6432
|
|
|
6433 -L <int> Sets the length of the seed substrings to align during multiseed alignment. Smaller values
|
|
|
6434 make alignment slower but more senstive. Default: the --sensitive preset of Bowtie 2 is
|
|
|
6435 used by default, which sets -L to 20. This option is only available for Bowtie 2 (for
|
|
|
6436 Bowtie 1 see -l).
|
|
|
6437
|
|
|
6438 --ignore-quals When calculating a mismatch penalty, always consider the quality value at the mismatched
|
|
|
6439 position to be the highest possible, regardless of the actual value. I.e. input is treated
|
|
|
6440 as though all quality values are high. This is also the default behavior when the input
|
|
|
6441 doesn't specify quality values (e.g. in -f mode). This option is invariable and on by default.
|
|
|
6442
|
|
|
6443
|
|
|
6444 Bowtie 2 paired-end options:
|
|
|
6445
|
|
|
6446 --no-mixed This option disables Bowtie 2's behavior to try to find alignments for the individual mates if
|
|
|
6447 it cannot find a concordant or discordant alignment for a pair. This option is invariable and
|
|
|
6448 and on by default.
|
|
|
6449
|
|
|
6450 --no-discordant Normally, Bowtie 2 looks for discordant alignments if it cannot find any concordant alignments.
|
|
|
6451 A discordant alignment is an alignment where both mates align uniquely, but that does not
|
|
|
6452 satisfy the paired-end constraints (--fr/--rf/--ff, -I, -X). This option disables that behavior
|
|
|
6453 and it is on by default.
|
|
|
6454
|
|
|
6455
|
|
|
6456 Bowtie 2 effort options:
|
|
|
6457
|
|
|
6458 -D <int> Up to <int> consecutive seed extension attempts can "fail" before Bowtie 2 moves on, using
|
|
|
6459 the alignments found so far. A seed extension "fails" if it does not yield a new best or a
|
|
|
6460 new second-best alignment. Default: 15.
|
|
|
6461
|
|
|
6462 -R <int> <int> is the maximum number of times Bowtie 2 will "re-seed" reads with repetitive seeds.
|
|
|
6463 When "re-seeding," Bowtie 2 simply chooses a new set of reads (same length, same number of
|
|
|
6464 mismatches allowed) at different offsets and searches for more alignments. A read is considered
|
|
|
6465 to have repetitive seeds if the total number of seed hits divided by the number of seeds
|
|
|
6466 that aligned at least once is greater than 300. Default: 2.
|
|
|
6467
|
|
|
6468 Bowtie 2 parallelization options:
|
|
|
6469
|
|
|
6470
|
|
|
6471 -p NTHREADS Launch NTHREADS parallel search threads (default: 1). Threads will run on separate processors/cores
|
|
|
6472 and synchronize when parsing reads and outputting alignments. Searching for alignments is highly
|
|
|
6473 parallel, and speedup is close to linear. Increasing -p increases Bowtie 2's memory footprint.
|
|
|
6474 E.g. when aligning to a human genome index, increasing -p from 1 to 8 increases the memory footprint
|
|
|
6475 by a few hundred megabytes. This option is only available if bowtie is linked with the pthreads
|
|
|
6476 library (i.e. if BOWTIE_PTHREADS=0 is not specified at build time). In addition, this option will
|
|
|
6477 automatically use the option '--reorder', which guarantees that output SAM records are printed in
|
|
|
6478 an order corresponding to the order of the reads in the original input file, even when -p is set
|
|
|
6479 greater than 1 (Bismark requires the Bowtie 2 output to be this way). Specifying --reorder and
|
|
|
6480 setting -p greater than 1 causes Bowtie 2 to run somewhat slower and use somewhat more memory then
|
|
|
6481 if --reorder were not specified. Has no effect if -p is set to 1, since output order will naturally
|
|
|
6482 correspond to input order in that case.
|
|
|
6483
|
|
|
6484 Bowtie 2 Scoring options:
|
|
|
6485
|
|
|
6486 --score_min <func> Sets a function governing the minimum alignment score needed for an alignment to be considered
|
|
|
6487 "valid" (i.e. good enough to report). This is a function of read length. For instance, specifying
|
|
|
6488 L,0,-0.2 sets the minimum-score function f to f(x) = 0 + -0.2 * x, where x is the read length.
|
|
|
6489 See also: setting function options at http://bowtie-bio.sourceforge.net/bowtie2. The default is
|
|
|
6490 L,0,-0.2.
|
|
|
6491
|
|
|
6492
|
|
|
6493 Bowtie 2 Reporting options:
|
|
|
6494
|
|
|
6495 -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
|
|
|
6496 deprecated. It will be removed in subsequent versions. What used to be called -M mode is still the
|
|
|
6497 default mode, but adjusting the -M setting is deprecated. Use the -D and -R options to adjust the
|
|
|
6498 effort expended to find valid alignments.
|
|
|
6499
|
|
|
6500 For reference, this used to be the old (now deprecated) description of -M:
|
|
|
6501 Bowtie 2 searches for at most <int>+1 distinct, valid alignments for each read. The search terminates when it
|
|
|
6502 can't find more distinct valid alignments, or when it finds <int>+1 distinct alignments, whichever
|
|
|
6503 happens first. Only the best alignment is reported. Information from the other alignments is used to
|
|
|
6504 estimate mapping quality and to set SAM optional fields, such as AS:i and XS:i. Increasing -M makes
|
|
|
6505 Bowtie 2 slower, but increases the likelihood that it will pick the correct alignment for a read that
|
|
|
6506 aligns many places. For reads that have more than <int>+1 distinct, valid alignments, Bowtie 2 does not
|
|
|
6507 guarantee that the alignment reported is the best possible in terms of alignment score. -M is
|
|
|
6508 always used and its default value is set to 10.
|
|
|
6509
|
|
|
6510
|
|
|
6511 'VANILLA' Bismark OUTPUT:
|
|
|
6512
|
|
|
6513 Single-end output format (tab-separated):
|
|
|
6514
|
|
|
6515 (1) <seq-ID>
|
|
|
6516 (2) <read alignment strand>
|
|
|
6517 (3) <chromosome>
|
|
|
6518 (4) <start position>
|
|
|
6519 (5) <end position>
|
|
|
6520 (6) <observed bisulfite sequence>
|
|
|
6521 (7) <equivalent genomic sequence>
|
|
|
6522 (8) <methylation call>
|
|
|
6523 (9) <read conversion
|
|
|
6524 (10) <genome conversion>
|
|
|
6525 (11) <read quality score (Phred33)>
|
|
|
6526
|
|
|
6527
|
|
|
6528 Paired-end output format (tab-separated):
|
|
|
6529 (1) <seq-ID>
|
|
|
6530 (2) <read 1 alignment strand>
|
|
|
6531 (3) <chromosome>
|
|
|
6532 (4) <start position>
|
|
|
6533 (5) <end position>
|
|
|
6534 (6) <observed bisulfite sequence 1>
|
|
|
6535 (7) <equivalent genomic sequence 1>
|
|
|
6536 (8) <methylation call 1>
|
|
|
6537 (9) <observed bisulfite sequence 2>
|
|
|
6538 (10) <equivalent genomic sequence 2>
|
|
|
6539 (11) <methylation call 2>
|
|
|
6540 (12) <read 1 conversion
|
|
|
6541 (13) <genome conversion>
|
|
|
6542 (14) <read 1 quality score (Phred33)>
|
|
|
6543 (15) <read 2 quality score (Phred33)>
|
|
|
6544
|
|
|
6545
|
|
|
6546 Bismark SAM OUTPUT (default):
|
|
|
6547
|
|
|
6548 (1) QNAME (seq-ID)
|
|
|
6549 (2) FLAG (this flag tries to take the strand a bisulfite read originated from into account (this is different from ordinary DNA alignment flags!))
|
|
|
6550 (3) RNAME (chromosome)
|
|
|
6551 (4) POS (start position)
|
|
|
6552 (5) MAPQ (always 255)
|
|
|
6553 (6) CIGAR
|
|
|
6554 (7) RNEXT
|
|
|
6555 (8) PNEXT
|
|
|
6556 (9) TLEN
|
|
|
6557 (10) SEQ
|
|
|
6558 (11) QUAL (Phred33 scale)
|
|
|
6559 (12) NM-tag (edit distance to the reference)
|
|
|
6560 (13) XX-tag (base-by-base mismatches to the reference. This does not include indels)
|
|
|
6561 (14) XM-tag (methylation call string)
|
|
|
6562 (15) XR-tag (read conversion state for the alignment)
|
|
|
6563 (16) XG-tag (genome conversion state for the alignment)
|
|
|
6564
|
|
|
6565 Each read of paired-end alignments is written out in a separate line in the above format.
|
|
|
6566
|
|
|
6567
|
|
|
6568 This script was last edited on 31 July 2012.
|
|
|
6569
|
|
|
6570 HOW_TO
|
|
|
6571 }
|
