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18
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1 #!/usr/bin/perl -w
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2 #Filename:
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3 #Author: Tian Dongmei
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4 #Email: tiandm@big.ac.cn
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5 #Date: 2013/7/19
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6 #Modified:
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7 #Description:
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8 my $version=1.00;
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9
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10 use File::Path;
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11 use strict;
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12 use File::Basename;
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13 #use Getopt::Std;
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14 use Getopt::Long;
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15 use RNA;
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16
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17 my %opts;
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18 GetOptions(\%opts,"r=s","p=s","m=s","mis:i","t:i","e:i","f:i","tag:s","o=s","time:s","h");
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19 if (!(defined $opts{r} and defined $opts{p} and defined $opts{m} and defined $opts{o} ) || defined $opts{h}) { #necessary arguments
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20 &usage;
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21 }
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22
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23 my $read=$opts{'r'};
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24 my $pre=$opts{'p'};
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25 my $mature=$opts{'m'};
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26
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27 my $dir=$opts{'o'};
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28 unless ($dir=~/\/$/) {$dir .="/";}
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29 if (not -d $dir) {
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30 mkdir $dir;
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31 }
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32
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33 my $threads=defined $opts{'t'} ? $opts{'t'} : 1;
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34 my $mismatch=defined $opts{'mis'} ? $opts{'mis'} : 0;
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35
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36 my $upstream = 2;
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37 my $downstream = 5;
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38
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39 $upstream = $opts{'e'} if(defined $opts{'e'});
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40 $downstream = $opts{'f'} if(defined $opts{'f'});
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41
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42 my $marks=defined $opts{'tag'} ? $opts{'tag'} : "";
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43
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44 my $time=Time();
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45 if (defined $opts{'time'}) { $time=$opts{'time'};}
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46
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47 my $tmpdir="${dir}/miRNA_Express_${time}";
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48 if(not -d $tmpdir){
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49 mkdir($tmpdir);
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50 }
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51 chdir $tmpdir;
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52
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53 `cp $pre ./`;
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54 my $pre_file_name=basename($pre);
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55
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56 &mapping(); # matures align to precursors && reads align to precursors;
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57
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58 my %pre_mature; # $pre_mature{pre_id}{matre_ID}{"mature"}[0]->start; $pre_mature{pre_id}{matre_ID}{"mature"}[1]->end;
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59 &maturePosOnPre(); # acknowledge mature positions on precursor
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60
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61 my %pre_read;
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62 &readPosOnPre(); # acknowledge reads positions on precursors
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63
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64 if(!(defined $opts{'tag'})){
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65 foreach my $key (keys %pre_read) {
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66 $pre_read{$key}[0][0]=~/:([\d|_]+)_x(\d+)$/;
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67 my @ss=split/_/,$1;
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68 for (my $i=1;$i<=@ss;$i++) {
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69 $marks .="Smp$i;";
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70 }
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71 last;
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72 }
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73 }
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74
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75 my %pre;## read in precursor sequences #$pre{pre_id}="CGTA...."
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76 &attachPre();
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77
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78 my $preno=scalar (keys %pre);
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79 print "Total Precursor Number is $preno !!!!\n";
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80
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81 my %struc; #mature star loop; $struc{$key}{"struc"}=$str; $struc{$key}{"mfe"}=$mfe;
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82 &structure();
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83
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84
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85 ##### analysis and print out && moRs
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86 my $aln=$dir."known_microRNA_express.aln";
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87 my $list=$dir."known_microRNA_express.txt";
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88 my $moRs=$dir."known_microRNA_express.moRs";
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89
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90 system("ln $mature $dir/known_microRNA_mature.fa ");
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91 system("ln $pre $dir/known_microRNA_precursor.fa ");
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92
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93 open ALN,">$aln";
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94 open LIST,">$list";
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95 open MORS,">$moRs";
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96
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97 $"="\t"; ##### @array print in \t
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98
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99 my @marks=split/\;/,$marks;
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100 #print LIST "#matueID\tpreID\tpos1\tpos2\tmatureExp\tstarExp\ttotalExp\n";
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101 print LIST "#matueID\tpreID\tpos1\tpos2";
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102 for (my $i=0;$i<@marks;$i++) {
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103 print LIST "\t",$marks[$i],"_matureExp";
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104 }
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105 for (my $i=0;$i<@marks;$i++) {
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106 print LIST "\t",$marks[$i],"_starExp";
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107 }
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108 for (my $i=0;$i<@marks;$i++) {
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109 print LIST "\t",$marks[$i],"_totalExp";
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110 }
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111 print LIST "\n";
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112 print ALN "#>precursor ID \n#precursor sequence\n#precursor structure (mfe)\n#RNA_seq\t@marks\ttotal\n";
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113 print MORS "#>precursor ID\tstrand\texpress_reads\texpress_reads\/total_reads\tblock_number\tprecursor_sequence\n#\tblock_start\tblock_end\t@marks\ttotal\ttag_number\tsequence\n";
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114 my %moRs;
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115
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116 foreach my $key (keys %pre) {
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117 print ALN ">$key\n$pre{$key}\n$struc{$key}{struc} ($struc{$key}{mfe})\n";
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118 next if(! (exists $pre_read{$key}));
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119 my @array=@{$pre_read{$key}};
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120 @array=sort{$a->[3]<=> $b->[3]} @array;
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121
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122 my $length=length($pre{$key});
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123
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124 my $maxline=-1;my $max=0; ### storage the maxinum express read line
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125 my $totalReadsNo=0;
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126 my @not_over=(); ### new read format better for moRs analysis
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127
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128 ####print out Aln file start
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129 for (my $i=0;$i<@array;$i++) {
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130 my $maps=$array[$i][3]+1;
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131 my $mape=$array[$i][3]+length($array[$i][4]);
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132 my $str="";
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133 $str .= "." x ($maps-1);
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134 $str .=$array[$i][4];
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135 $str .="." x ($length-$mape);
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136 $str .=" ";
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137
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138 $array[$i][0]=~/:([\d|_]+)_x(\d+)$/;
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139 my @sample=split /\_/,$1;
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140 my $total=$2;
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141 print ALN $str,"@sample","\t",$total,"\n";
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142
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143 if($total>$max){$max=$total; $maxline=$i;}
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144 $totalReadsNo+=$total;
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145
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146 push @not_over,[$key,$maps,$mape,$array[$i][0],$total,"+"];
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147 }
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148 ####print out Aln file end
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149
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150 #### express list start
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151 my ($ms,$me,$ss,$se);
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152 if (!(exists($pre_mature{$key}))) {
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153 $ms=$array[$maxline][3]+1;
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154 $me=$array[$maxline][3]+length($array[$maxline][4]);
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155 ($ss,$se)=&other_pair($ms,$me,$struc{$key}{'struc'});
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156
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157 my ($mexp,$sexp,$texp)=&express($ms-$upstream,$me+$downstream,$ss-$upstream,$se+$downstream,\@array);
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158 print LIST "$key\t$key\tmature:$ms..$me\tstar:$ss..$se\t@$mexp\t@$sexp\t@$texp\n";
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159 }
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160 else{
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161 foreach my $maID (keys %{$pre_mature{$key}}) {
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162 $ms=$pre_mature{$key}{$maID}{"mature"}[0];
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163 $me=$pre_mature{$key}{$maID}{"mature"}[1];
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164 $ss=$pre_mature{$key}{$maID}{"star"}[0];
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165 $se=$pre_mature{$key}{$maID}{"star"}[1];
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166 my ($mexp,$sexp,$texp)=&express($ms-$upstream,$me+$downstream,$ss-$upstream,$se+$downstream,\@array);
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167 print LIST "$maID\t$key\tmature:$ms..$me\tstar:$ss..$se\t@$mexp\t@$sexp\t@$texp\n";
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168 }
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169 }
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170 #### express list end
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171
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172 #### analysis moRs start
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173 my @result; my @m_texp;my $m_texp=0; ### moRs informations
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174
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175 while (@not_over>0) {
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176 my @over=@not_over;
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177 @not_over=();
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178
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179 #·á¶È×î¸ßtag
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180 my $m_max=0;my $m_maxline=-1;my $m_start=0;my $m_end=0;my $m_exp=0;my @m_exp;my $m_no=1;
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181 for (my $i=0;$i<@over;$i++) {
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182 my @m_array=@{$over[$i]};
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183 if ($m_max<$m_array[4]) {
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184 $m_max=$m_array[4];
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185 $m_maxline=$i;
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186 }
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187 }
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188 $m_start=$over[$m_maxline][1];
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189 $m_end=$over[$m_maxline][2];
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190 $m_exp=$m_max;
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191 $over[$m_maxline][3]=~/:([\d|_]+)_x(\d+)$/;
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192 my @m_nums=split/_/,$1;
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193 for (my $j=0;$j<@m_nums;$j++) {
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194 $m_exp[$j]=$m_nums[$j];
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195 }
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196
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197 #ͳ¼ÆÒÔ·á¶È×î¸ßtagΪ×ø±êµÄreads, Á½¶ËλÖòîÒì²»³¬¹ý3nt
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198 for (my $i=0;$i<@over;$i++) {
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199 next if($i==$m_maxline);
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200 my @m_array=@{$over[$i]};
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201 if (abs($m_array[1]-$m_start)<=3 && abs($m_array[2]-$m_end)<=3) {
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202 $m_exp+=$m_array[4];
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203 $m_no++;
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204 $m_array[3]=~/:([\d|_]+)_x(\d+)$/;
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205 my @m_nums=split/_/,$1;
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206 for (my $j=0;$j<@m_nums;$j++) {
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207 $m_exp[$j] +=$m_nums[$j];
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208 }
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209 }
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210 elsif($m_array[1]>=$m_end || $m_array[2]<=$m_start){push @not_over,[@{$over[$i]}];} #È¥³ý¿çÔ½blockµÄreads
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211 }
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212 if($m_exp>5){### 5¸öreads
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213 $m_texp+=$m_exp;
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214 for (my $j=0;$j<@m_exp;$j++) {
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215 $m_texp[$j]+=$m_exp[$j];
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216 }
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217 my $string=&subseq($pre{$key},$m_start,$m_end,"+");
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218 push @result,"\t$m_start\t$m_end\t@m_exp\t$m_exp\t$m_no\t$string" ;
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219 }
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220 }
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221
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222 my $str=scalar @result;
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223 my $percent=sprintf("%.2f",$m_texp/$totalReadsNo);
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224 $str=">$key\t+\t$m_texp\t$percent\t".$str."\t$pre{$key}";
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225 @{$moRs{$str}}=@result;
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226
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227 #### analysis moRs end
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228 }
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229
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230 ##### moRs print out start
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231 foreach my $key (keys %moRs) {
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232 my @tmp=split/\t/,$key;
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233 next if ($tmp[4]<=2);
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234 next if($tmp[3]<0.95);
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235 my @over;
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236 for (my $i=0;$i<@{$moRs{$key}};$i++) {
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237 my @arrayi=split/\t/,$moRs{$key}[$i];
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238 for (my $j=0;$j<@{$moRs{$key}};$j++) {
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239 next if($i==$j);
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240 my @arrayj=split/\t/,$moRs{$key}[$j];
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241 if ((($arrayj[1]-$arrayi[2]>=0 && $arrayj[1]-$arrayi[2] <=3) || ($arrayj[1]-$arrayi[2]>=18 && $arrayj[1]-$arrayi[2] <=25) )||(($arrayi[1]-$arrayj[2]>=0 && $arrayi[1]-$arrayj[2] <=3)||($arrayi[1]-$arrayj[2]>=18 && $arrayi[1]-$arrayj[2] <=25))) {
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242 push @over,$moRs{$key}[$i];
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243 }
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244 }
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245 }
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246 if (@over>0) {
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247 print MORS "$key\n";
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248 foreach (@{$moRs{$key}}) {
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249 print MORS "$_\n";
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250 }
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251 }
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252 }
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253 ###### moRs print out end
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254 close ALN;
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255 close LIST;
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256 close MORS;
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257
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258 $"=" ";##### reset
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259
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260
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261 ################### Sub programs #################
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262 sub express{
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263 my ($ms,$me,$ss,$se,$read)=@_;
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264 my (@mexp,@sexp,@texp);
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265 $$read[0][0]=~/:([_|\d]+)_x(\d+)$/;
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266 my @numsample=split/_/,$1;
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267 for (my $i=0;$i<@numsample;$i++) {
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268 $mexp[$i]=0;
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269 $sexp[$i]=0;
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270 $texp[$i]=0;
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271 }
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272
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273 for (my $i=0;$i<@{$read};$i++) {
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274 my $start=$$read[$i][3]+1;
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275 my $end=$$read[$i][3]+length($$read[$i][4]);
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276 $$read[$i][0]=~/:([_|\d]+)_x(\d+)$/;
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277 my $expresses=$1;
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278 my @nums=split/_/,$expresses;
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279
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280 for (my $j=0;$j<@nums;$j++) {
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281 $texp[$j]+=$nums[$j];
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282 }
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283 if ($start>=$ms && $end<=$me) {
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284 for (my $j=0;$j<@nums;$j++) {
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285 $mexp[$j]+=$nums[$j];
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286 }
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287 }
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288 if ($start>=$ss && $end<=$se) {
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289 for (my $j=0;$j<@nums;$j++) {
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290 $sexp[$j]+=$nums[$j];
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291 }
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292 }
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293 }
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294 return(\@mexp,\@sexp,\@texp);
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295 }
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296
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297 sub structure{
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298 foreach my $key (keys %pre_mature) {
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299 if (!(defined $pre{$key})){die "!!!!! No precursor sequence $key, please check it!\n";}
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300 my ($str,$mfe)=RNA::fold($pre{$key});
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301 $struc{$key}{"struc"}=$str;
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302 $struc{$key}{"mfe"}=sprintf ("%.2f",$mfe);
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303
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304 foreach my $id (keys %{$pre_mature{$key}}) {
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305 ($pre_mature{$key}{$id}{"star"}[0],$pre_mature{$key}{$id}{"star"}[1])=&other_pair($pre_mature{$key}{$id}{"mature"}[0],$pre_mature{$key}{$id}{"mature"}[1],$str);
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306 }
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307 =cut
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308 ##### Nucleotide complementary
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309 my @tmp=split//,$str;
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310 my %a2b;
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311 my @bps;
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312 for (my $i=0;$i<@tmp;$i++) {
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313 if ($tmp[$i] eq "("){push @bps,$i+1 ; next;}
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314 if ($tmp[$i] eq ")") {
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315 my $up=pop @bps;
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316 $a2b{$i+1}=$up;
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317 $a2b{$up}=$i+1;
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318 }
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319 }
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320
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321 ##### search star position
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322 foreach my $id (keys %{$pre_mature{$key}}) {
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323 my $n=0;
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324 for (my $i=$pre_mature{$key}{$id}{"mature"}[0];$i<=$pre_mature{$key}{$id}{"mature"}[1] ; $i++) {
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325 if (defined $a2b{$i}) {
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326 my $a=$i; my $b=$a2b{$i};
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327 if($a>$b){
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328 $pre_mature{$key}{$id}{"star"}[0]=$b-$n+2;
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329 $pre_mature{$key}{$id}{"star"}[1]=$b-$n+2+($pre_mature{$key}{$id}{"mature"}[1]-$pre_mature{$key}{$id}{"mature"}[0]);
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330 }
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331 if($a<$b{
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332 $pre_mature{$key}{$id}{"star"}[1]=$b+$n+2;
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333 $pre_mature{$key}{$id}{"star"}[0]=$b+$n+2-($pre_mature{$key}{$id}{"mature"}[1]-$pre_mature{$key}{$id}{"mature"}[0]);
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334 }
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335 last;
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336 }
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337 $n++;
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338 }
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339 }
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340 =cut
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341 }
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342 }
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343 sub other_pair{
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344 my ($start,$end,$structure)=@_;
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345 ##### Nucleotide complementary
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346 my @tmp=split//,$structure;
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347 my %a2b; my @bps;
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348 for (my $i=0;$i<@tmp;$i++) {
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349 if ($tmp[$i] eq "("){push @bps,$i+1 ; next;}
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350 if ($tmp[$i] eq ")") {
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351 my $up=pop @bps;
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352 $a2b{$i+1}=$up;
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353 $a2b{$up}=$i+1;
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354 }
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355 }
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356 ##### search star position
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357 my $n=0;my $startpos; my $endpos;
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358 for (my $i=$start;$i<=$end ; $i++) {
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359 if (defined $a2b{$i}) {
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360 my $a=$i; my $b=$a2b{$i};
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361 # if($a>$b){
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362 # $startpos=$b-$n+2;
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363 # $endpos=$b-$n+2+($end-$start);
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364 # }
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365 # if($a<$b){
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366 $endpos=$b+$n+2;
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367 if($endpos>length($structure)){$endpos=length($structure);}
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368 $startpos=$b+$n+2-($end-$start);
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369 if($startpos<1){$startpos=1;}
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370 # }
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371 last;
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372 }
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373 $n++;
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374 }
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375 return ($startpos,$endpos);
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376 }
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377 sub attachPre{
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378 open IN, "<$pre_file_name";
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379 my $name;
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380 while (my $aline=<IN>) {
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381 chomp $aline;
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382 if ($aline=~/^>(\S+)/) {
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383 $name=$1;
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384 next;
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385 }
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386 $pre{$name} .=$aline;
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387 }
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388 close IN;
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389 }
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390 sub readPosOnPre{
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391 open IN,"<read_mapped.bwt";
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392 while (my $aline=<IN>) {
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393 chomp $aline;
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394 my @tmp=split/\t/,$aline;
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395 my $id=lc($tmp[2]);
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396 push @{$pre_read{$tmp[2]}},[@tmp];
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397 }
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398 close IN;
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399 }
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400 sub maturePosOnPre{
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401 open IN,"<mature_mapped.bwt";
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402 while (my $aline=<IN>) {
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403 chomp $aline;
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|
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404 my @tmp=split/\t/,$aline;
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|
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405 my $mm=$tmp[0];
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|
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406 # $mm=~s/\-3P|\-5P//i;
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|
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407 $mm=lc($mm);
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|
|
408 my $pm=$tmp[2];
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|
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409 $pm=lc($pm);
|
|
|
410
|
|
|
411 # next if ($mm ne $pm);### stringent mapping let7a only allowed to map pre-let7a
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|
|
412 next if($mm!~/$pm/);
|
|
|
413 # print "$tmp[2]\t$tmp[0]\n";
|
|
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414 # $pre_mature{$tmp[2]}{$tmp[0]}{"mature"}[0]=$tmp[3]-$upstream;
|
|
|
415 # $pre_mature{$tmp[2]}{$tmp[0]}{"mature"}[0]=0 if($pre_mature{$tmp[2]}{$tmp[0]}{"mature"}[0]<0);
|
|
|
416 # $pre_mature{$tmp[2]}{$tmp[0]}{"mature"}[1]=$tmp[3]+length($tmp[4])-1+$downstream;
|
|
|
417 $pre_mature{$tmp[2]}{$tmp[0]}{"mature"}[0]=$tmp[3]+1;
|
|
|
418 $pre_mature{$tmp[2]}{$tmp[0]}{"mature"}[1]=$tmp[3]+length($tmp[4]);
|
|
|
419 }
|
|
|
420 close IN;
|
|
|
421 }
|
|
|
422 sub mapping{
|
|
|
423 my $err;
|
|
|
424 ## build bowtie index
|
|
|
425 print STDERR "building bowtie index\n";
|
|
|
426 $err = `bowtie-build $pre_file_name miRNA_precursor`;
|
|
|
427
|
|
|
428 ## map mature sequences against precursors
|
|
|
429 print STDERR "mapping mature sequences against index\n";
|
|
|
430 $err = `bowtie -p $threads -f -v 0 -a --best --strata --norc miRNA_precursor $mature mature_mapped.bwt`;
|
|
|
431
|
|
|
432 ## map reads against precursors
|
|
|
433 print STDERR "mapping read sequences against index\n";
|
|
|
434 $err=`bowtie -p $threads -f -v $mismatch -a --best --strata --norc miRNA_precursor $read --al mirbase_mapped.fa --un mirbase_not_mapped.fa read_mapped.bwt `;
|
|
|
435
|
|
|
436 }
|
|
|
437
|
|
|
438 sub subseq{
|
|
|
439 my $seq=shift;
|
|
|
440 my $beg=shift;
|
|
|
441 my $end=shift;
|
|
|
442 my $strand=shift;
|
|
|
443
|
|
|
444 my $subseq=substr($seq,$beg-1,$end-$beg+1);
|
|
|
445 if ($strand eq "-") {
|
|
|
446 $subseq=revcom($subseq);
|
|
|
447 }
|
|
|
448 return uc $subseq;
|
|
|
449 }
|
|
|
450
|
|
|
451 sub revcom{
|
|
|
452 my $seq=shift;
|
|
|
453 $seq=~tr/ATCGatcg/TAGCtagc/;
|
|
|
454 $seq=reverse $seq;
|
|
|
455 return uc $seq;
|
|
|
456 }
|
|
|
457
|
|
|
458 sub Time{
|
|
|
459 my $time=time();
|
|
|
460 my ($sec,$min,$hour,$day,$month,$year) = (localtime($time))[0,1,2,3,4,5,6];
|
|
|
461 $month++;
|
|
|
462 $year+=1900;
|
|
|
463 if (length($sec) == 1) {$sec = "0"."$sec";}
|
|
|
464 if (length($min) == 1) {$min = "0"."$min";}
|
|
|
465 if (length($hour) == 1) {$hour = "0"."$hour";}
|
|
|
466 if (length($day) == 1) {$day = "0"."$day";}
|
|
|
467 if (length($month) == 1) {$month = "0"."$month";}
|
|
|
468 #print "$year-$month-$day $hour:$min:$sec\n";
|
|
|
469 return("$year-$month-$day-$hour-$min-$sec");
|
|
|
470 }
|
|
|
471
|
|
|
472 sub usage{
|
|
|
473 print <<"USAGE";
|
|
|
474 Version $version
|
|
|
475 Usage:
|
|
|
476 $0 -r -p -m -mis -t -e -f -tag -o -time
|
|
|
477 mandatory parameters:
|
|
|
478 -p precursor.fa miRNA precursor sequences from miRBase # must be absolute path
|
|
|
479 -m mature.fa miRNA sequences from miRBase # must be absolute path
|
|
|
480 -r reads.fa your read sequences #must be absolute path
|
|
|
481
|
|
|
482 -o output directory
|
|
|
483
|
|
|
484 options:
|
|
|
485 -mis [int] number of allowed mismatches when mapping reads to precursors, default 0
|
|
|
486 -t [int] threads number,default 1
|
|
|
487 -e [int] number of nucleotides upstream of the mature sequence to consider, default 2
|
|
|
488 -f [int] number of nucleotides downstream of the mature sequence to consider, default 5
|
|
|
489 -tag [string] sample marks# eg. sampleA;sampleB;sampleC
|
|
|
490 -time sting #make directory time,default is the local time
|
|
|
491 -h help
|
|
|
492 USAGE
|
|
|
493 exit(1);
|
|
|
494 }
|
|
|
495
|
