3
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1 #!/usr/bin/perl
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2
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3 #######
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4 # POD #
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5 #######
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6
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7 =pod
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8
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9 =head1 NAME
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10
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11 ecoli_mlst.pl 25-10-2011
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12
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13 =head1 SYNOPSIS
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14
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15 C<perl ecoli_mlst.pl -a fas -g fasta>
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16
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17 =head1 DESCRIPTION
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18
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19 The script searches for multilocus sequence type (MLST) alleles in
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20 I<E. coli> genomes according to Mark Achtman's scheme with seven
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21 house-keeping genes (I<adk>, I<fumC>, I<gyrB>, I<icd>, I<mdh>,
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22 I<purA>, and I<recA>) [Wirth et al., 2006]. I<NUCmer> from the
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23 L<I<MUMmer package>|http://mummer.sourceforge.net/> is used to
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24 compare the given allele sequences to bacterial genomes via
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25 nucleotide alignments.
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26
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27 Download the allele files (adk.fas ...) and the sequence type file
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28 ('publicSTs.txt') from this website:
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29 http://mlst.warwick.ac.uk/mlst/dbs/Ecoli
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30
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31 To run C<ecoli_mlst.pl> include all I<E. coli> genome files (file
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32 extension e.g. 'fasta'), all allele sequence files (file extension
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33 'fas') and 'publicSTs.txt' in the current working directory. The
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34 allele profiles are parsed from the created *.coord files and written
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35 to a result file, plus additional information from the file
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36 'publicSTs.txt'. Also, the corresponding allele sequences (obtained
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37 from the allele input files) are concatenated for each I<E. coli>
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38 genome into a result multi-fasta file. Option B<-c> can be used to
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39 initiate an alignment for this multi-fasta file with
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40 L<I<ClustalW>|http://www.clustal.org/clustal2/> (standard alignment
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41 parameters; has to be in the C<$PATH> or change variable
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42 C<$clustal_call>). The alignment fasta output file can be used
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43 directly for L<I<RAxML>|http://sco.h-its.org/exelixis/web/software/raxml/index.html>. CAREFUL the Phylip alignment format from
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44 I<ClustalW> allows only 10 characters per strain ID.
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45
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46 C<ecoli_mlst.pl> works with complete and draft genomes. However,
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47 several genomes cannot be included in a single input file!
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48
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49 Obviously, only for those genomes whose allele sequences have been
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50 deposited in Achtman's allele database results can be obtained. If an
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51 allele is not found in a genome it is marked by a '?' in the result
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52 profile file and a place holder 'XXX' in the result fasta file. For
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53 these cases a manual I<NUCmer> or I<BLASTN> might be useful to fill the
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54 gaps and L<C<run_sub_seq.pl>|/run_sub_seq> to get the corresponding
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55 'new' allele sequences.
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56
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57 Non-NCBI fasta headers for the genome files have to have a unique ID
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58 directly following the '>' (e.g. 'Sakai', '55989' ...).
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59
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60 =head1 OPTIONS
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61
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62 =head2 Mandatory options
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63
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64 =over 22
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65
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66 =item B<-a>=I<str>, B<-alleles>=I<str>
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67
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68 File extension of the MLST allele fasta files, e.g. 'fas' (<=> B<-g>).
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69
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70 =item B<-g>=I<str>, B<-genomes>=I<str>
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71
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72 File extension of the I<E. coli> genome fasta files, e.g. 'fasta' (<=> B<-a>).
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73
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74 =back
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75
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76 =head2 Optional options
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77
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78 =over
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79
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80 =item B<-h>, B<-help>
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81
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82 Help (perldoc POD)
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83
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84 =item B<-c>, B<-clustalw>
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85
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86 Call L<I<ClustalW>|http://www.clustal.org/clustal2/> for alignment
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87
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88 =back
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89
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90 =head1 OUTPUT
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91
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92 =over 17
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93
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94 =item F<ecoli_mlst_profile.txt>
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95
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96 Tab-separated allele profiles for the I<E. coli> genomes, plus additional info from 'publicSTs.txt'
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97
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98 =item F<ecoli_mlst_seq.fasta>
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99
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100 Multi-fasta file of all concatenated allele sequences for each genome
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101
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102 =item F<*.coord>
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103
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104 Text files that contain the coordinates of the I<NUCmer> hits for each genome and allele
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105
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106 =item (F<errors.txt>)
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107
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108 Error file, summarizing number of not found alleles or unclear I<NUCmer> hits
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109
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110 =item (F<ecoli_mlst_seq_aln.fasta>)
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111
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112 Optional, L<I<ClustalW>|http://www.clustal.org/clustal2/> alignment in Phylip format
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113
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114 =item (F<ecoli_mlst_seq_aln.dnd>)
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115
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116 Optional, I<ClustalW> alignment guide tree
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117
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118 =back
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119
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120 =head1 EXAMPLE
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121
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122 =over
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123
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124 =item C<perl ecoli_mlst.pl -a fas -g fasta -c>
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125
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126 =back
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127
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128 =head1 VERSION
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129
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130 0.3 update: 30-01-2013
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131
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132 =head1 AUTHOR
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133
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134 Andreas Leimbach aleimba[at]gmx[dot]de
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135
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136 =head1 LICENSE
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137
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138 This program is free software: you can redistribute it and/or modify
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139 it under the terms of the GNU General Public License as published by
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140 the Free Software Foundation; either version 3 (GPLv3) of the License,
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141 or (at your option) any later version.
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142
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143 This program is distributed in the hope that it will be useful, but
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144 WITHOUT ANY WARRANTY; without even the implied warranty of
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145 MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the GNU
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146 General Public License for more details.
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147
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148 You should have received a copy of the GNU General Public License
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149 along with this program. If not, see L<http://www.gnu.org/licenses/>.
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150
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151 =cut
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152
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153
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154 ########
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155 # MAIN #
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156 ########
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157
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158 use strict;
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159 use warnings;
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160 use Getopt::Long; # module to get options from the command line
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161
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162
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163 ### Get the options with Getopt::Long, works also abbreviated and with two "--": -g, --g, -genomes ...
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164 my $usage = "perldoc $0";
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165 die system($usage) unless @ARGV;
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166 my $allele_ext = ''; # file extension of allele fasta files (<=> $genome_ext)
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167 my $genome_ext = ''; # file extension of E. coli genome fasta files (<=> allele_ext)
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168 my $clustalw = ''; # optionally, call ClustalW for alignment
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169 my $help = ''; # run perldoc on the POD
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170 GetOptions ('alleles=s' => \$allele_ext, 'genomes=s' => \$genome_ext, 'clustalw' => \$clustalw, 'help' => \$help);
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171
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172
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173 ### Run perldoc on POD if no arguments or help
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174 if (!$genome_ext || !$allele_ext) {
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175 die system($usage);
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176 } elsif ($help) {
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177 die system($usage);
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178 }
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179
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180
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181 ### Check if result files already exist, overwrite or exit script after user question
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182 my $ecoli_allele_seq = 'ecoli_mlst_seq.fasta'; # multi-fasta result file with concatenated allele seqs for each E. coli genome (sequences are from the MLST allele files)
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183 my $clustal_aln = 'ecoli_mlst_seq_aln.fasta'; # fasta alignment file from optional clustalW alignment
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184 if (-e $ecoli_allele_seq) {
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185 print "A previous analysis exists, overwrite the old result files [y|n]? ";
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186 my $stdin = <STDIN>;
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187 chomp $stdin;
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188 if ($stdin =~ /n/i) {
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189 die "Script abborted!\n\n";
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190 } elsif ($stdin =~ /y/i) {
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191 if (-e $clustal_aln) { # get rid of the optional clustalW alignment fasta-file before program run
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192 unlink $clustal_aln;
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193 }
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194 }
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195 }
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196
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197
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198 ### The MLST alleles from the Achtman scheme
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199 my @alleles = ('adk', 'fumC', 'gyrB', 'icd', 'mdh', 'purA', 'recA');
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200
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201
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202 ### Read directory and look for corresponding files
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203 my $dirname = ".";
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204 my @genome_files; # array to save all the fasta genome files
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205 my %allele_files; # hash to save all the multi-fasta allele files
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206 opendir(DIR, $dirname) or die "Can't opendir $dirname: $!\n";
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207 while (defined(my $file = readdir(DIR))) { # go through each file in the directory
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208 if ($file eq 'ecoli_mlst_seq.fasta') { # don't use the result file from a previous analysis, the allele multi fasta file 'ecoli_mlst_multi.fasta' (see below)!
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209 next;
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210 } elsif ($file =~ /^(\S+)\.$genome_ext$/ && (-s $file < 9000000)) { # don't use files > 10 MB, E. coli fasta file shouldn't be too big (normally around 5 MB), otherwise probably wrong fasta file in folder
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211 push (@genome_files, $file);
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212 }
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213 foreach my $allele (@alleles) {
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214 if ($file =~ /^$allele\S*\.$allele_ext$/i) {
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215 $allele_files{$allele} = $file;
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216 }
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217 }
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218 }
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219 closedir(DIR);
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220 die "No E. coli genome fasta-files were found!\n" unless scalar @genome_files; # empty array in scalar context returns 0
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221 die "No allele fasta-files were found!\n" unless scalar %allele_files; # empty hash in scalar context returns 0
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222
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223
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224 ### Look for multi-fasta genome files and concatenate them to a single-fasta entry (e.g. draft genomes). Subsequently concatenate all E. coli genome sequence files to one multi-fasta/-genome file for the subsequent nucmer run. Additionally parse and associate the E. coli accession#s/unique IDs and descriptions (strain names ...) from the genomes
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225 my $genome_file = 'ecoli_multi.fasta'; # multi-genome/-fasta file for the nucmer run, used as a temp file, will be deleted at the end of the script
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226 open(GENOMES, ">$genome_file") or die "Failed to create file '$genome_file': $!\n";
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227 my $genome_number = 0; # used to control if lines in *.coords files correspond to the overall genome count
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228 my %genome_desc; # hash to associate accession#/unique ID with genome descriptions
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229 foreach my $file (@genome_files) {
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230 $genome_number++;
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231 open(MULTI, "<$file") or die "Failed to open E. coli genome file '$file': $!\n";
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232 my @multi = <MULTI>; # read the whole genome file (potential multi-fasta file)
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233 my @IDs = grep(/^>/, @multi); # get ID lines in fasta file
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234 if (scalar @IDs > 1) { # multi-fasta files
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235 my $new_id; # new ID line for the multi-fasta file
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236 foreach (@IDs) { # discard plasmid ID lines in complete genomes for new file (name with chromosome ID line)
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237 if (!/plasmid/) {
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238 chomp;
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239 $new_id = $_;
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240 last; # jump out of the loop if a non-plasmid ID found
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241 }
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242 }
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243 if (!defined($new_id)) {
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244 die "The file '$file' only contains plasmids, program exits!\n";
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245 }
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246 my ($acc, $desc) = acc_desc($new_id); # subroutine to fill hash %genome_desc with acc#/unique ID and genome description
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247 print GENOMES ">$acc $desc; artificial genome\n"; # print the now shortened ID-line for the new single-fasta entry in the result multi-genome file
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248 foreach (@multi) { # print the rest of the new single-fasta file
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249 if (/^>/) { # skip ID lines of the old multi-fasta file
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250 next;
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251 }
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252 print GENOMES;
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253 }
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254 } elsif (scalar @IDs == 1) { # non-multi-fasta files
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255 my ($acc, $desc) = acc_desc($IDs[0]);
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256 print GENOMES ">$acc $desc\n"; # print the new shortened ID-line
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257 foreach (@multi) {
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258 if (/^>/) { # skip ID line, since new one already printed
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259 next;
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260 }
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261 print GENOMES;
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262 }
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263 } else { # wrong fasta files
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264 die "File '$file' does not include a fasta ID line, program exits!\n";
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265 }
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266 print GENOMES "\n";
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267 close MULTI;
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268 }
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269 close GENOMES;
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270 my @delete; # files to be deleted after program run
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271 if (-e $genome_file) {
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272 push (@delete, $genome_file);
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273 } else {
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274 die "The multi-fasta E. coli genome file \'$genome_file\' could not be created for the NUCmer run: $!\n";
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275 }
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276
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277
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278 ### Parse file 'publicSTs.txt' to get additional info for each sequence type
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279 my $st_file = 'publicSTs.txt';
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280 open(ST, "<$st_file") or die "Failed to open sequence type file '$st_file': $!\n";
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281 my %seq_type; # hash to save ST info to each allele profile
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282 my $header = <ST>; # get rid of file header
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283 while (<ST>) {
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284 chomp;
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285 my @st = split (/\t/, $_);
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286 my $profile = "$st[3] $st[4] $st[5] $st[6] $st[7] $st[8] $st[9]"; # allele profile
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287 my $info = "$st[0]\t$st[1]\t$st[2]\t$st[10]\t$st[11]"; # associated info to allele profile
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288 # $st[0] = ST, 1 = ST_COMPLEX, 2 = ANCESTRAL_GROUP, 3-9 = adk-recA alleles, 10 = SOURCE, 11 = REFSTRAIN
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289 $seq_type{$profile} = $info;
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290 }
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291 close ST;
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292
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293
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294 ### Run nucmer with allele sequences as REFERENCES and the created 'ecoli_multi.fasta' file as QUERY
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295 my @created_files; # used to print out files that are created at the end of the script
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296 my %coord_files;
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297 foreach (keys %allele_files) {
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298 system ("nucmer --prefix $_-all_ecoli $allele_files{$_} $genome_file"); # system call; seperate args not possible, probably because nucmer not a system command?!
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299 system ("show-coords -lT $_-all_ecoli.delta > $_-all_ecoli.coords"); # '-l' include seq length in output, '-T' tab-separated output
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300 if (-e "$_-all_ecoli.coords") {
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301 $coord_files{$_} = "$_-all_ecoli.coords";
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302 push (@delete, "$_-all_ecoli.delta");
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303 push (@created_files, "\t\t\t$_-all_ecoli.coords\n");
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304 } else {
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305 die "Coord file '$_-all_ecoli.coords' could not be created: $!\n";
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306 }
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307 }
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308
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309
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310 ### Parse *.coords nucmer result files for MLST alleles and corresponding accession#s
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311 my @summary_err; # array to store error summary for error file
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312 my @detail_err; # array to store the more detailed errors for error file
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313 my $error = 0; # switches to '1' if an error is detected
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314 my %strain_allele; # declare hash, that's subsequently used as 'hash in hash' in sub parse_coords to associate MLST alleles and accession#s
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315 foreach (sort keys %coord_files) {
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316 parse_coord($coord_files{$_}, $_); # subroutine to fill %strain_allele
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317 }
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318
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319
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320 ### Print the corresponding allele sequences and allele profile (plus additional info) for each E. coli genome
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321 open(SEQ, ">$ecoli_allele_seq") or die "Failed to create file '$ecoli_allele_seq': $!\n";
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322 my $ecoli_allele_profile = 'ecoli_mlst_profile.txt'; # tab-separated result file for the allele profile of each E. coli genome plus additional info from file 'publicSTs.txt' from the Achtman MLST DB
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323 open(PROFILE, ">$ecoli_allele_profile") or die "Failed to create file '$ecoli_allele_profile': $!\n";
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324 print PROFILE "Strain"; # header for profile file
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325 foreach (sort @alleles) {
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326 print PROFILE "\t$_";
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327 }
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328 print PROFILE "\tST\tST_COMPLEX\tANCESTRAL_GROUP\tSOURCE\tREFSTRAIN\n"; # info from file 'publicSTs.txt'
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329 foreach my $acc (sort {lc $genome_desc{$a} cmp lc $genome_desc{$b}} keys %genome_desc) { # call hash %genome_desc by keys (acc#s), but sort by values (genome desc.s) of the hash case-insensitively (all in lowercase, because Perl sorts lowercase after uppercase!)
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330 print SEQ ">$genome_desc{$acc} $acc\n"; # print fasta ID line for 'ecoli_mlst_seq.fasta'
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331 print PROFILE "$genome_desc{$acc}"; # print genome desc for strain in first column for 'ecoli_mlst_profile.txt'
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332 my $profile = ''; # allele profile to extract info from %seq_type
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333 foreach my $allele (sort keys %strain_allele) {
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334 if (defined $strain_allele{$allele}->{$acc}) {
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335 open(ALLELE, "<$allele_files{$allele}") or die "Failed to open allele file $allele_files{$allele}: $!\n";
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336 while (my $line = <ALLELE>) { # search for corresponding allele in multi-fasta allele file
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337 chomp $line;
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338 if ($line =~ /^>$strain_allele{$allele}->{$acc}$/i) {
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339 $line = <ALLELE>; # don't need the ID line but the following seq lines
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340 while ($line !~ /^>/) { # print sequence in result file until another '>' is found
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341 chomp $line;
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342 print SEQ "$line";
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343 $line = <ALLELE>;
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344 }
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345 }
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346 }
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347 close ALLELE;
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348 $strain_allele{$allele}->{$acc} =~ s/^(\D+)(\d+)$/$2/; # only keep allele number
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349 print PROFILE "\t$strain_allele{$allele}->{$acc}";
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350 $profile .= "$strain_allele{$allele}->{$acc} "; # concat allele numbers in $profile to extract info from %seq_type
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351 } else {
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352 print SEQ "XXX"; # place holder if allele of genome not determined
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353 print PROFILE "\t?"; # place holder as well
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354 unshift (@detail_err, "$genome_desc{$acc} didn't give a hit with $allele, marked by \'XXX\' in allele sequences and \'?\' in allele profile!\n");
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355 next;
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356 }
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357 }
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358 print SEQ "\n\n"; # blank line in front of each ID line
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359 chop $profile; # get rid of the last space, so it can be used as the key in %seq_type
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360 if (defined $seq_type{$profile}) { # print ST and additional info from file 'publicST.txt' in 'ecoli_mlst_profile.txt'
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361 print PROFILE "\t$seq_type{$profile}\n";
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362 } else {
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363 print PROFILE "\t?\t?\t?\t?\t?\n";
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364 }
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365 }
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366 close SEQ;
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367 close PROFILE;
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368 if (-e $ecoli_allele_seq && $ecoli_allele_profile) { # push new created files in array for print out
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369 push (@created_files, "\n\tResult files:\n\t\t\t$ecoli_allele_seq\t-> Allele sequences!\n", "\t\t\t$ecoli_allele_profile\t-> Allele profile plus info from 'publicSTs.txt'!\n");
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370 } else {
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371 die "The result files $ecoli_allele_seq and $ecoli_allele_profile could not be created: $!\n";
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372 }
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373
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374
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375 ### Print errors in error file,
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376 if ($error == 1) { # error(s) occurred
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377 my $err_file = 'errors.txt';
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378 open(ERR, ">$err_file") or die "Failed to create file '$err_file': $!\n";
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379 print ERR "Error summary:\n";
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380 print ERR @summary_err; # filled in sub 'parse_coords'
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381 print ERR "\nDetailed error output:\n";
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382 print ERR @detail_err;
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383 push (@created_files, "\t\t\t$err_file\t\t-> Error file!\n");
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384 close ERR;
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385 }
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386
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387
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388 ### Delete unneeded files, temp file 'ecoli_multi.fasta' (see above) and the *.delta files from the NUCmer run
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389 foreach my $goners (@delete) {
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390 unlink $goners or warn "Could not remove file '$goners': $!";
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391 }
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392
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393
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394 ### Print newly created files
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395 print "\n###########################################################################\n";
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396 print "The following files were created:\n";
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397 print "\tCoordinates of MLST alleles in each genome:\n";
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398 print @created_files;
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399 print "\n###########################################################################\n";
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400
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401
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402 ### Align with ClustalW if option '-c' is given
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403 if ($clustalw) {
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404 print "\nStarting ClustalW alignment with file $ecoli_allele_seq ...";
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405 my $out = $ecoli_allele_seq;
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406 $out =~ s/\.fasta$//;
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407 my $clustal_call = "clustalw -infile=$ecoli_allele_seq -outfile=$clustal_aln -align -type=DNA -output=phylip -tree -newtree=$out\_aln.dnd -outputtree=phylip";
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408 system ($clustal_call);
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409 }
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410
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411 exit;
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412
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413
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414 ###############
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415 # Subroutines #
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416 ###############
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417
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418 ### Subroutine to associate the acc# to the genome description in hash %genome_desc (see above)
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419 sub acc_desc {
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420 my $ID = shift;
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421 chomp $ID;
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422 if ($ID =~ /^>gi\|\d+\|(emb|gb|dbj|ref)\|(\w+\d+\.\d)\|\s(.+)$/) { # NCBI fasta header
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423 # e.g.: >gi|387605479|ref|NC_017626.1| Escherichia coli 042, complete genome
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424 # $1 = DB (embl, genbank, ddbj, refseq), $2 = acc#, $3 = genome description
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425 my $desc = shorten_desc($3); # subroutine to shorten the genome description
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426 $genome_desc{$2} = $desc; # associate accession# with genome description
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427 return ($2, $desc);
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428 } elsif ($ID =~ /^>(\S+)\s*(\S*)\s*(.*)$/) { # headers after EMBOSS's union of multi-fasta files, and other headers with a unique ID after '>'
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429 # e.g.: >NC_011748.1 NC_011748.1 Escherichia coli 55989 chromosome, complete genome
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|
430 # $1 = acc#, $2 = genome desc for drafts|duplicated acc# for complete genomes, $3 = genome desc for complete genomes
|
|
431 my $desc = $2 . ' ' . $3;
|
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432 if ($1 eq $2) { # complete genomes have acc# double after EMBOSS's union (see above)
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433 $desc = $3;
|
|
434 }
|
|
435 $desc = shorten_desc($desc);
|
|
436 $genome_desc{$1} = $desc;
|
|
437 return ($1, $desc);
|
|
438 }
|
|
439 return 0;
|
|
440 }
|
|
441
|
|
442
|
|
443 ### Subroutine to parse *.coord nucmer result files for MLST alleles and corresponding accession#s
|
|
444 sub parse_coord {
|
|
445 my ($coord_file, $allele) = @_;
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|
446 my $lines = 0; # lines of respective *.coords file (without header); control if hit is missing or insensitive hits are present in comparison to $genome_number for the error file
|
|
447 my $discarded = 0; # number of discarded hits/lines in coord file for error file
|
|
448 open (COORD, "$coord_file") or die "Failed to open file '$coord_file': $!\n";
|
|
449 while (<COORD>) {
|
|
450 chomp;
|
|
451 if ( /\d+\t\d+\t\d+\t\d+\t(\d+)\t(\d+)\t(\d+\.\d+)\t(\d+)\t\d+\t(\D+\d+)\t(\w*\d+\.\d)$/ ) {
|
|
452 # since the ID lines of the fastas were shortened in temp file 'ecoli_multi.fasta', the acc#s should be the last element of each line in the coords file
|
|
453 # $1 = length of ref alignment, $2 = length of query alignment, $3 = identity percentage,
|
|
454 # $4 = length of ref seq, $5 = allele-nr. (e.g. ADK15), $6 = accession-nr.
|
|
455 $lines++; # after 'if' to skip headers
|
|
456 if ($1 != $4) { # write error to @detail_err for error file
|
|
457 push (@detail_err, "$genome_desc{$6}, $5: Reference (allele) alignment doesn't have a correct length, therefore allele is not included in output!\n");
|
|
458 $discarded++;
|
|
459 next;
|
|
460 } elsif ($2 != $4) {
|
|
461 push (@detail_err, "$genome_desc{$6}, $5: Query (genome) alignment doesn't have a correct length, therefore allele is not included in output!\n");
|
|
462 $discarded++;
|
|
463 next;
|
|
464 } elsif ($3 != '100.00') {
|
|
465 push (@detail_err, "$genome_desc{$6}, $5: Identity is not 100\%, therefore is not included in output!\n");
|
|
466 $discarded++;
|
|
467 next;
|
|
468 }
|
|
469 $strain_allele{$allele}{$6} = $5; # associate allele# with acc# and store as hash in hash in %strain_allele
|
|
470 }
|
|
471 }
|
|
472 close COORD;
|
|
473 # error identifications for later print out in error.txt
|
|
474 if ($discarded == 0) {
|
|
475 if ($lines < $genome_number) {
|
|
476 $error = 1; # switch $error from 0 to 1 to indicate error was found
|
|
477 push (@summary_err, $genome_number - $lines, " $allele allele(s) missing!\n");
|
|
478 }
|
|
479 } elsif ($discarded > 0) {
|
|
480 $error = 1;
|
|
481 if (($lines - $discarded) == $genome_number) {
|
|
482 push (@summary_err, "Total number of specific assigned $allele alleles is correct, but $discarded non-specific hit(s) discarded!\n");
|
|
483 } elsif (($lines - $discarded) < $genome_number) {
|
|
484 push (@summary_err, $genome_number - ($lines - $discarded), " $allele allele(s) missing and $discarded non-specific hit(s)!\n");
|
|
485 }
|
|
486 }
|
|
487 return 1;
|
|
488 }
|
|
489
|
|
490
|
|
491 ### Shorten the genome descriptions of the ID headers
|
|
492 sub shorten_desc {
|
|
493 my $desc = shift;
|
|
494 $desc =~ s/Escherichia coli/Ecoli/;
|
|
495 $desc =~ s/\'//g;
|
|
496 $desc =~ s/( DNA, complete genome| chromosome, complete genome|, complete genome| chromosome, complete sequence| complete genome|, complete sequence|, strain (\S+)|, whole genome shotgun sequence)$//;
|
|
497 $desc =~ s/\s/_/g;
|
|
498 return $desc;
|
|
499 }
|