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1 #!/usr/bin/perl -w
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2 my $version=1.00;
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3 use strict;
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4 use warnings;
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5 use Getopt::Long;
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6 use Getopt::Std;
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7 use threads;
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8 use threads::shared;
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9 use Parallel::ForkManager;
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10 use lib '/leofs/biotrans/chentt/perl_module/';
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11 #perl ../siRNA.pl -i config -g /leofs/biotrans/projects/rice/smallRNA/sRNA_package/bin/test/ref/genome.fa -f /share_bio/hs4/disk3-4/Reference/Plants/Rice_TIGR/Reference/TIGR/version_6.1/all.dir/all.gff3 -path /leofs/biotrans/projects/rice/smallRNA/sRNA_package/bin/ -o /leofs/biotrans/projects/rice/smallRNA/sRNA_package/bin/test -t 3 -rfam /leofs/biotrans/projects/rice/smallRNA/sRNA_package/bin/test/ref/Rfam.fasta -idx /leofs/biotrans/projects/rice/smallRNA/sRNA_package/bin/test/ref/genome -idx2 /leofs/biotrans/projects/rice/smallRNA/sRNA_package/bin/test/ref/rfam -deg deg -n 25 -nat class/nat_1 -repeat class/repeat_1 -cen centromere_TIGR.txt -format fastq
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12 print "
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13 #####################################
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14 # #
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15 # sRNA cluster #
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16 # #
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17 #####################################
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18 ";
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19 ###########################################################################################
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20 my $usage="$0
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21 Options:
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22 -i input file# raw data file
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23 -tag string #raw data sample name
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24 -g genome file
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25 -f gff file
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26
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27 -o workdir file
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28 -path script path
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29 -t int, number of threads [1]
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30 -format fastq, fq, fasta or fa
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31 -idx string, genome file index, file-prefix #(must be indexed by bowtie-build) The parameter
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32 string must be the prefix of the bowtie index. For instance, if
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33 the first indexed file is called 'h_sapiens_37_asm.1.ebwt' then
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34 the prefix is 'h_sapiens_37_asm'.##can be null
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35 -mis int number of allowed mismatches when mapping reads to genome, default 0
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36 -rfam string, input file# rfam database file.
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37 -idx2 string, rfam file index, file-prefix #(must be indexed by bowtie-build) The parameter
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38 string must be the prefix of the bowtie index. For instance, if
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39 the first indexed file is called 'h_sapiens_37_asm.1.ebwt' then
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40 the prefix is 'h_sapiens_37_asm'.##can be null
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41
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42 -v int report end-to-end hits w/ <=v mismatches; ignore qualities,default 0; used in rfam alignment
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43
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44 -a string, ADAPTER string. default is ATCTCGTATG.
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45 -n int max hits number,default 25; used in genome alignment
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46 -d int distance of tag to merged a cluster; default 100
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47 -p cluster method F :conventional default is F
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48 T :NIBLES
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49 -l int the length of the upstream and downstream,default 1000;used in position annotate
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50
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51 -nat natural antisense transcripts file
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52 -repeat repeat information file out of Repeatmasker
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53 -deg file config of de sample
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54 -cen centromere file input
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55 -span plot span, default 50000
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56 ";
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57
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58 my %options;
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59 GetOptions(\%options,"i:s@","tag:s@","g=s","phed:i","f=s","o=s","a:s","path:s","p=s","format=s","nat:s","repeat:s","deg:s","n:i","mis:i","rfam:s","t:i","v:i","d:i","l:i","idx:s","idx2:s","cen:s","span:s","h");
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60 #print help if that option is used
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61 if($options{h}){die $usage;}
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62
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63 my @filein=@{$options{'i'}};
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64 my @mark=@{$options{'tag'}};
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65
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66 #my $config=$options{'i'};
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67 my $genome_fa=$options{'g'};
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68 my $gff=$options{'f'};
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69
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70
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71 ##########################################################################################
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72 my $predir=`pwd`;
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73 chomp $predir;
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74 my $workdir=defined($options{'o'}) ? $options{'o'}:$predir;
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75
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76 my $path=$options{'path'};
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77
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78 my $t=defined($options{'t'})? $options{'t'}:1; #threads number
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79
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80 my $mis=defined $options{'mis'} ? $options{'mis'}:0;
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81
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82 my $mis_rfam=defined $options{'v'} ? $options{'v'}:0;
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83
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84 my $hit=defined $options{'n'}?$options{'n'}:25;
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85
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86 my $distance_of_merged_tag=defined $options{'d'} ? $options{'d'}:100;
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87
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88 my $up_down_dis=defined $options{'l'} ?$options{'l'}:1000;
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89
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90 my $cluster_mothod=defined $options{'p'}?$options{'p'}:"F";
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91
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92 my $format=$options{'format'};
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93 #if ($format ne "fastq" && $format ne "fq" && $format ne "fasta" && $format ne "fa") {
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94 # die "Parameter \"-format\" is error! Parameter is fastq, fq, fasta or fa\n";
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95 #}
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96
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97 my $adpter="ATCTCGTATG"; #adapter
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98 if (defined $options{'a'}) {$adpter=$options{'a'};}
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99
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100
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101 my $phred_qv=64;
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102 if(defined $opts{'phred'}){$phred_qv=$opts{'phred'};}
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103 my $sample_number;
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104 my ($dir,$dir_tmp);
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105 ################################ MAIN ##################################################
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106 print "\ncluster program start:";
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107 my $time=Time();
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108 make_dir_tmp();
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109
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110 my @clip;
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111 my $mark;
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112 my $sample_mark;
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113
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114 my $config=$dir."/input_config";
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115 open CONFIG,">$config";
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116 for (my $i=0;$i<@filein;$i++) {
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117 print CONFIG $filein[$i],"\t",$mark[$i],"\n";
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118 }
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119 close CONFIG;
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120 if (@filein != @mark) {
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121 die "Maybe config file have some wrong!!!\n";
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122 }
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123 $sample_number=@mark;
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124 $mark=join "\t",@mark;
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125 $sample_mark=join "\#",@mark;
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126
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127
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128 #read_config();
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129
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130 trim_adapter_and_filter();
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131
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132 my $filter_out=$dir."preProcess\/"."collapse_reads_out.fa";## raw clean data
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133 my $data2=$filter_out; ### mirbase not mapped reads
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134 my $data3=$dir."\/rfam_match\/rfam_not_mapped\.fa"; ### rfam not mapped reads
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135 my $bed=$dir."cluster\/"."sample\.bed";
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136 my $read=$dir."cluster\/"."sample_reads\.cluster";
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137 my $read_txt=$dir."cluster\/"."cluster\.txt";
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138 my $rpkm=$dir."cluster\/"."sample_rpkm\.cluster";
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139 my $preprocess;
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140 my $cluster_file;
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141 my $annotate_dir;
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142 my $deg_dir;
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143 my $plot_dir;
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144 my %id;
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145 for (my $i=0;$i<@mark ;$i++) {
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146 $id{$mark[$i]}=$i+4;
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147 }
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148
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149 print "\n######## tiandm test start ###########\n";
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150 print "\@mark: @mark\n\%id keys number:";
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151 print scalar keys %id;
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152 print "\n";
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153 foreach my $kyess (keys %id){
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154 print $kyess," --> $id{$kyess}\n";
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155 }
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156 print "\n######## tiandm test end ############\n";
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157 group_and_filter(); #collapse reads to tags
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158
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159 rfam();
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160
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161 my @map_read;
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162 my $map_tag=0;
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163 genome();
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164
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165 bwt2bed();
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166
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167 cluster();
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168
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169 quantify();
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170
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171 phase();
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172
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173 if (defined($options{'nat'})&&defined($options{'repeat'})) {
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174 class();
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175 }
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176 else{
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177 get_genelist();
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178 }
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179
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180 annotate();
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181
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182 genome_length();
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183
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184 plot();
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185
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186 my @pairdir;
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187 if (defined($options{'deg'})) {
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188 dec();
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189 infor_merge();
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190 }
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191 else{infor_merge_no_dec()}
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192 html();
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193 print "\ncluster program end:";
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194 Time();
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195 ############################sub program###################################################
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196 sub make_dir_tmp{
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197
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198 #make temporary directory
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199 if(not -d "$workdir\/cluster_runs"){
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200 mkdir("$workdir\/cluster_runs");
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201 mkdir("$workdir\/cluster_runs\/ref\/");
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202 }
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203
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204 $dir="$workdir\/cluster_runs\/";
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205 #print STDERR "mkdir $dir\n\n";
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206 return;
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207 }
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208
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209 #sub read_config{
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210 # open IN,"<$config";
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211 # while (my $aline=<IN>) {
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212 # chomp $aline;
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213 # my @tmp=split/\t/,$aline;
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214 # push @filein,$tmp[0];
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215 # push @mark,$tmp[1];
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216 # }
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217 # close IN;
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218 # if (@filein != @mark) {
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219 # die "Maybe config file have some wrong!!!\n";
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220 # }
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221 # $sample_number=@mark;
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222 # $mark=join "\t",@mark;
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223 # $sample_mark=join "\#",@mark;
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224 #}
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225
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226
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227 sub trim_adapter_and_filter{
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228 my $time=time();
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229 $preprocess=$dir."preProcess/";
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230 mkdir $preprocess;
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231 my $can_use_threads = eval 'use threads; 1';
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232 if ($can_use_threads) {
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233 # Do processing using threads
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234 my @filein1=@filein; my @mark1=@mark;
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235 while (@filein1>0) {
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236 my @thrs; my @res;
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237 for (my $i=0;$i<$t ;$i++) {
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238 last if(@filein1==0);
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239 my $in=shift @filein1;
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240 my $out=shift @mark1;
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241 push @clip,$dir."preProcess\/$out\_clip\.fq";
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242 $thrs[$i]=threads->create(\&clips,$in,$out);
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243 }
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244 for (my $i=0;$i<@thrs;$i++) {
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245 $res[$i]=$thrs[$i]->join();
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246 }
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247 }
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248 }
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249 else {
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250 # Do not processing using threads
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251 for (my $i=0;$i<@filein ;$i++) {
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252 my $in=$filein[$i];
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253 my $out=$mark[$i];
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254 push @clip,$dir."preProcess\/$out\_clip\.fq";
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255 &clips($in,$out);
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256 }
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257 }
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258 }
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259
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260 sub clips{
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261 my ($filein,$fileout)=@_;
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262 my $adapter=$dir."preProcess\/$fileout\_clip\.fq";
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263 if($format eq "fq" || $format eq "fastq"){
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264 my $clip=`fastx_clipper -a $adpter -M 6 -Q $phred_qv -i $filein -o $adapter`;
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265 }
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266 if($format eq "fa" || $format eq "fasta"){
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267 my $clip=`fastx_clipper -a $adpter -M 6 -i $filein -o $adapter`;
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268 }
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269 #my $clean=$dir."preProcess\/$fileout\_clean.fq";
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270 #my $filter=`filterReadsByLength.pl -i $adapter -o $clean -min 18 -max 40 `;
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271 return $fileout;
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272 }
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273
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274 sub group_and_filter{
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275 #my ($ins,$data)=@_;
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276 my @ins=@clip;
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277 my $str="";
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278 my $group_out_file=$dir."preProcess\/"."collapse_reads.fa";
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279 #print "$$ins[0]\t$$ins[0]\n";
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280 for (my $i=0;$i<@clip;$i++) {
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281 $str .="-i $clip[$i] ";
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282 #print "$$ins[$i]\n";
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283 }
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284 my $group=`perl $path\/collapseReads2Tags.pl $str -mark seq -o $group_out_file -format $format`;
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285 print "perl $path\/collapseReads2Tags.pl $str -mark seq -o $group_out_file -format $format\n\n";
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286
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287 my $l_out=$dir."preProcess\/"."collapse_reads_18-40.fa";
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288 my $tmpmark=join ",", @mark;
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289
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290 my $length_f=`perl $path\/filterReadsByLength_1.pl -i $group_out_file -o $l_out -min 18 -max 40 -mark $tmpmark`;
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291 print "perl $path\/filterReadsByLength_1.pl -i $group_out_file -o $l_out -min 18 -max 40 -mark $tmpmark\n\n";
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292 my $cout_f=`perl $path\/filterReadsByCount.pl -i $l_out -o $filter_out -mark $sample_mark`;
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293 print "perl $path\/filterReadsByCount.pl -i $l_out -o $filter_out -mark $sample_mark\n\n";
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294 my $plot_l_D=`perl $path/Length_Distibution.pl -i $dir/preProcess/reads_length_distribution_after_count_filter.txt -o $dir/preProcess/length.html `;
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295 print "perl $path\/Length_Distibution.pl -i $dir\/preProcess\/reads_length_distribution_after_count_filter.txt -o $dir\/preProcess\/length\.html\n\n";
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296 return 0;
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297 }
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298
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299 sub rfam{
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300 if (defined $options{'idx2'}) {
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301 system("perl $path\/rfam.pl -i $data2 -ref $options{rfam} -v $mis_rfam -p $t -o $dir -index $options{idx2}");
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302 }else{
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303 system("perl $path\/rfam.pl -i $data2 -ref $options{rfam} -v $mis_rfam -p $t -o $dir");
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304 }
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305 my $tag=join "\\;" ,@mark;
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306 my $rfam_count=`perl $path\/count_rfam_express.pl -i $dir\/rfam_match\/rfam_mapped.bwt -tag $tag -o $dir\/rfam_match\/rfam_non-miRNA_annotation.txt`;
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307 return 0;
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308 }
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309 sub genome{
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310 if(defined $options{'idx'}){
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311 system("perl $path\/matching.pl -i $data3 -g $genome_fa -v $mis -p $t -r $hit -o $dir -index $options{idx}") ;
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312 }else{
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313 system("perl $path\/matching.pl -i $data3 -g $genome_fa -v $mis -p $t -r $hit -o $dir ") ;
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314 }
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315 #=================== mapping sta ===================================================
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316 my $map_file=$dir."genome_match\/genome_mapped\.fa";
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317 open (MAP,"<$map_file")||die"$!";
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318 print "\n#each sample mapping reads sta:\n\n";
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319 print "#$mark\ttotal\n";
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320 while (my $ID=<MAP>) {
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321 chomp $ID;
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322 my @tmp=split/\:/,$ID;
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323 my @exp=split/\_/,$tmp[1];
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324 $exp[-1] =~ s/^x//;
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325 for (my $i=0;$i<@exp ;$i++) {
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326 $map_read[$i]+=$exp[$i];
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327 }
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328 $map_tag++;
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329 my $seq=<MAP>;
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330 }
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331 my $map_read=join"\t",@map_read;
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332 print "$map_read\n\n";
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333 print "#total mapped tags:$map_read\n\n";
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334 close MAP;
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335 return 0;
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336 }
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337
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338 sub bwt2bed{
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339 $cluster_file=$dir."cluster\/";
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340 mkdir ("$cluster_file");
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341 print "sam file changed to bed file\n";
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342 my ($file) = $dir."genome_match\/genome_mapped\.bwt";
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343
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344 my $sam2bed=`perl $path\/sam2Bed_bowtie.pl -i $file -mark $sample_mark -o $bed `;
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345 print "perl $path\/sam2Bed_bowtie.pl -i $file -mark $sample_mark -o $bed\n\n";
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346 return 0;
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347 }
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348
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349 sub cluster{
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350 print "tags is ready to merged clusters\n\n";
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351 my ($file) =$bed;
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352 if ($cluster_mothod eq "F") {
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353 my $cluster=`perl $path\/conventional.pl -i $file -d $distance_of_merged_tag -n $sample_number -mark $sample_mark -o $read -t $read_txt`;
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354 print "Using converntional method\n perl $path\/conventional.pl -i $file -d $distance_of_merged_tag -n $sample_number -mark $sample_mark -o $read -t $read_txt\n\n";
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355 }
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356 elsif($cluster_mothod eq "T"){
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357 my $cluster=`perl $path\/nibls.pl -f $file -m $distance_of_merged_tag -o $read -t $read_txt -k $sample_mark`;
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358 print "Using nibls method\n perl $path\/nibls.pl -f $file -m $distance_of_merged_tag -o $read -t $dir\/cluster.txt -k $sample_mark\n\n";
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359 }
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360 else{print "\-p is wrong!\n\n";}
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361 return 0;
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362 }
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363
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364
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365 sub quantify{
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366 print "clusters is ready to quantified\n\n";
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367 my @depth=@map_read;
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368 pop @depth;
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369 my $depth=join ",",@depth;
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370 my $quantify=`perl $path\/quantify.pl -i $read -d $depth -o $rpkm`;
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371 print "perl $path\/quantify_siRNA.pl -i $read -d $depth -o $rpkm\n\n\n";
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372 return 0;
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373 }
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374
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375 sub phase{
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376 $annotate_dir=$dir."annotate\/";
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377 mkdir ("$annotate_dir");
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378 print "clusters is to predict phase siRNA\n";
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379 my $phase=`perl $path\/phased_siRNA.pl -i $read_txt -o $annotate_dir\/phase.out`;
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380 print "perl $path\/phased_siRNA.pl -i $read_txt -o $annotate_dir\/phase.out\n\n\n";
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381 return 0;
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382 }
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383
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384 sub class{
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385 print "clusters is ready to annotate by sources\n\n";
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386 my $nat=$options{'nat'};
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387 my $repeat=$options{'repeat'};
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388 my $class=`perl $path\/ClassAnnotate.pl -i $rpkm -g $gff -n $nat -r $repeat -p $annotate_dir\/phase.out -o $annotate_dir\/sample_class.anno -t $annotate_dir\/nat.out -l $dir\/ref\/genelist.txt`;
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389 print "perl $path\/ClassAnnotate.pl -i $rpkm -g $gff -n $nat -r $repeat -p $annotate_dir\/phase.out -o $annotate_dir\/sample_class.anno -t $annotate_dir\/nat.out -l $dir\/ref\/genelist.txt\n\n";
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390 }
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391
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392 sub annotate{
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393 print "clusters is ready to annotate by gff file\n\n";
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394 my $file;
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395 if (defined($options{'nat'})&&defined($options{'repeat'})) {
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396 $file="$annotate_dir\/sample_class.anno";
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397 }
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398 else{
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399 $file=$rpkm;
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400 }
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401 my $annotate=`perl $path\/Annotate.pl -i $file -g $dir\/ref\/genelist.txt -d $up_down_dis -o $annotate_dir\/sample_c_p.anno`;
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402 print "perl $path\/Annotate.pl -i $file -g $dir\/ref\/genelist.txt -d $up_down_dis -o $annotate_dir\/sample_c_p.anno\n\n";
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403 return 0;
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404 }
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405 sub get_genelist{
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406
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407 my $get_genelist=`perl $path\/get_genelist.pl -i $gff -o $dir\/ref\/genelist.txt`;
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408 print "perl $path\/get_genelist.pl -i $gff -o $dir\/ref\/genelist.txt";
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409 }
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410
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411 sub dec{
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412 print "deg reading\n\n";
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413 my $deg_file=$options{'deg'};
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414 open IN,"<$deg_file";
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415 my @deg;
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416 my $s=0;
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417 while (my $aline=<IN>) {
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418 chomp $aline;
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419 next if($aline=~/^\#/);
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|
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420 $deg[$s]=$aline;
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|
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421 my @ea=split/\s+/,$aline;
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422 push @pairdir,"$ea[0]_VS_$ea[1]\/";
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423 #print "$deg[$s]\n";
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|
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424 $s++;
|
|
|
425 }
|
|
|
426 close IN;
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|
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427 $deg_dir=$dir."deg\/";
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|
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428 mkdir ("$deg_dir");
|
|
|
429 my $max_process = 10;
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|
|
430 my $pm = new Parallel::ForkManager( $max_process );
|
|
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431 my $number=@deg-1;
|
|
|
432 foreach(0..$number){
|
|
|
433 $pm->start and next;
|
|
|
434 &dec_pel($deg[$_]);
|
|
|
435 $pm->finish;
|
|
|
436 }
|
|
|
437 $pm->wait_all_children;
|
|
|
438 }
|
|
|
439
|
|
|
440 sub dec_pel{
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|
|
441 print "\n******************\nstart:\n";
|
|
|
442 Time();
|
|
|
443 my $sample=shift(@_);
|
|
|
444 my @each=split/\s+/,$sample;
|
|
|
445 print "$each[0]\t$each[1]\n";
|
|
|
446 my $deg_sample_dir=$deg_dir."$each[0]_VS_$each[1]\/";
|
|
|
447 mkdir ("$deg_sample_dir");
|
|
|
448 print "read: $read\n";
|
|
|
449 print "deg_sample_dir: $deg_sample_dir\n";
|
|
|
450 print "$id{$each[0]}\t$each[0]\n";
|
|
|
451 print "$id{$each[1]}\t$each[1]\n";
|
|
|
452 my $deg=`perl $path\/DEGseq_2.pl -i $read -outdir $deg_sample_dir -column1 $id{$each[0]} -mark1 $each[0] -column2 $id{$each[1]} -mark2 $each[1]`; #-depth1 -depth2
|
|
|
453 my $time2=time();
|
|
|
454 print "end:\n*************************\n";
|
|
|
455 Time();
|
|
|
456 sleep 1;
|
|
|
457 }
|
|
|
458
|
|
|
459 sub infor_merge{
|
|
|
460 my ($input,$mark);
|
|
|
461 foreach (@pairdir) {
|
|
|
462 print "@pairdir\n";
|
|
|
463 $mark.=" -mark $_ ";
|
|
|
464 $input.=" -i $dir/deg\/$_\/output_score\.txt ";
|
|
|
465 print "$input\n$mark\n";
|
|
|
466 }
|
|
|
467 my $infor_merge=`perl $path\/SampleDEGseqMerge.pl $input $mark -f $annotate_dir\/sample_c_p.anno -n $sample_number -o $dir\/total.result `;
|
|
|
468 print "perl $path\/SampleDEGseqMerge.pl $input $mark -f $annotate_dir\/sample_c_p.anno -n $sample_number -o $dir\/total.result\n\n";
|
|
|
469 }
|
|
|
470
|
|
|
471 sub infor_merge_no_dec{
|
|
|
472 my $infor_merge_no_dec=`cp $annotate_dir\/sample_c_p.anno $dir\/total.result`;
|
|
|
473 }
|
|
|
474
|
|
|
475 sub genome_length{
|
|
|
476 my $length=`perl $path\/count_ref_length.pl -i $genome_fa -o $dir\/ref\/genome\.length`;
|
|
|
477 print "perl $path\/count_ref_length.pl -i $genome_fa -o $dir\/ref\/genome\.length\n\n"
|
|
|
478
|
|
|
479 }
|
|
|
480
|
|
|
481 sub plot{
|
|
|
482 $plot_dir="$dir\/plot\/";
|
|
|
483 mkdir ("$plot_dir");
|
|
|
484 my $span=defined($options{span})?$options{span}:50000;
|
|
|
485 my $cen="";
|
|
|
486 if (defined $options{cen}) {
|
|
|
487 $cen="-cen $options{cen}";
|
|
|
488 }
|
|
|
489 my $plot=`perl $path/sRNA_plot.pl -c $rpkm -g $dir/ref/genelist.txt -span 50000 -mark $sample_mark -l $dir/ref/genome\.length $cen -o $plot_dir/cluster.html -out $plot_dir/cluster.txt `;
|
|
|
490 "print perl $path/sRNA_plot.pl -c $rpkm -g $dir/ref/genelist.txt -span 50000 -mark $sample_mark -l $dir/ref/genome.length $cen -o $plot_dir/cluster.html -out $plot_dir/cluster.txt \n";
|
|
|
491
|
|
|
492 }
|
|
|
493
|
|
|
494 sub html{
|
|
|
495 my $pathfile="$dir/path.txt";
|
|
|
496 open PA,">$pathfile";
|
|
|
497 print PA "$config\n";
|
|
|
498 print PA "$preprocess\n";
|
|
|
499 print PA "$dir"."rfam_match\n";
|
|
|
500 print PA "$dir"."genome_match\n";
|
|
|
501 print PA "$cluster_file\n";
|
|
|
502 print PA "$annotate_dir\n";
|
|
|
503 print PA "$plot_dir\n";
|
|
|
504 if (defined($deg_dir)) {
|
|
|
505 print PA "$deg_dir\n";
|
|
|
506 }
|
|
|
507 close PA;
|
|
|
508 my $html=`perl $path\/html.pl -i $pathfile -format $format -o $dir/result.html`;
|
|
|
509 }
|
|
|
510
|
|
|
511 sub Time{
|
|
|
512 my $time=time();
|
|
|
513 my ($sec,$min,$hour,$day,$month,$year) = (localtime($time))[0,1,2,3,4,5,6];
|
|
|
514 $month++;
|
|
|
515 $year+=1900;
|
|
|
516 if (length($sec) == 1) {$sec = "0"."$sec";}
|
|
|
517 if (length($min) == 1) {$min = "0"."$min";}
|
|
|
518 if (length($hour) == 1) {$hour = "0"."$hour";}
|
|
|
519 if (length($day) == 1) {$day = "0"."$day";}
|
|
|
520 if (length($month) == 1) {$month = "0"."$month";}
|
|
|
521 print "$year-$month-$day $hour:$min:$sec\n";
|
|
|
522 return("$year-$month-$day-$hour-$min-$sec");
|
|
|
523 }
|
|
|
524 #################################################################################
|
