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comparison quantify.pl @ 50:7b5a48b972e9 draft

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author big-tiandm
date Fri, 05 Dec 2014 00:11:02 -0500
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49:f008ab2cadc6 50:7b5a48b972e9
1 #!/usr/bin/perl -w
2 #Filename:
3 #Author: Tian Dongmei
4 #Email: tiandm@big.ac.cn
5 #Date: 2013/7/19
6 #Modified:
7 #Description:
8 my $version=1.00;
9
10 use File::Path;
11 use strict;
12 use File::Basename;
13 #use Getopt::Std;
14 use Getopt::Long;
15 #use RNA;
16
17 my %opts;
18 GetOptions(\%opts,"r=s","p=s","m=s","mis:i","t:i","e:i","f:i","tag:s","o=s","h");
19 if (!(defined $opts{r} and defined $opts{p} and defined $opts{m} and defined $opts{o} ) || defined $opts{h}) { #necessary arguments
20 &usage;
21 }
22
23 my $read=$opts{'r'};
24 my $pre=$opts{'p'};
25 my $mature=$opts{'m'};
26
27 my $dir=$opts{'o'};
28 unless ($dir=~/\/$/) {$dir .="/";}
29 if (not -d $dir) {
30 mkdir $dir;
31 }
32
33 my $threads=defined $opts{'t'} ? $opts{'t'} : 1;
34 my $mismatch=defined $opts{'mis'} ? $opts{'mis'} : 0;
35
36 my $upstream = 2;
37 my $downstream = 5;
38
39 $upstream = $opts{'e'} if(defined $opts{'e'});
40 $downstream = $opts{'f'} if(defined $opts{'f'});
41
42 my $marks=defined $opts{'tag'} ? $opts{'tag'} : "";
43
44 my $time=Time();
45
46 my $tmpdir="${dir}/known_miRNA_Express";
47 if(not -d $tmpdir){
48 mkdir($tmpdir);
49 }
50 chdir $tmpdir;
51
52 `cp $pre ./`;
53 my $pre_file_name=basename($pre);
54
55 &mapping(); # matures align to precursors && reads align to precursors;
56
57 my %pre_mature; # $pre_mature{pre_id}{matre_ID}{"mature"}[0]->start; $pre_mature{pre_id}{matre_ID}{"mature"}[1]->end;
58 &maturePosOnPre(); # acknowledge mature positions on precursor
59
60 my %pre_read;
61 &readPosOnPre(); # acknowledge reads positions on precursors
62
63 if(!(defined $opts{'tag'})){
64 foreach my $key (keys %pre_read) {
65 $pre_read{$key}[0][0]=~/:([\d|_]+)_x(\d+)$/;
66 my @ss=split/_/,$1;
67 for (my $i=1;$i<=@ss;$i++) {
68 $marks .="Smp$i;";
69 }
70 last;
71 }
72 }
73
74 my %pre;## read in precursor sequences #$pre{pre_id}="CGTA...."
75 &attachPre();
76
77 my $preno=scalar (keys %pre);
78 print "Total Precursor Number is $preno !!!!\n";
79
80 my %struc; #mature star loop; $struc{$key}{"struc"}=$str; $struc{$key}{"mfe"}=$mfe;
81 &structure();
82
83
84 ##### analysis and print out && moRs
85 my $aln=$dir."known_microRNA_express.aln";
86 my $list=$dir."known_microRNA_express.txt";
87 my $moRs=$dir."known_microRNA_express.moRs";
88
89 system("ln -s $mature $dir/known_microRNA_mature.fa ");
90 system("ln -s $pre $dir/known_microRNA_precursor.fa ");
91
92 open ALN,">$aln";
93 open LIST,">$list";
94 open MORS,">$moRs";
95
96 $"="\t"; ##### @array print in \t
97
98 my @marks=split/\;/,$marks;
99 #print LIST "#matueID\tpreID\tpos1\tpos2\tmatureExp\tstarExp\ttotalExp\n";
100 print LIST "#matueID\tpreID\tpos1\tpos2";
101 for (my $i=0;$i<@marks;$i++) {
102 print LIST "\t",$marks[$i],"_matureExp";
103 }
104 for (my $i=0;$i<@marks;$i++) {
105 print LIST "\t",$marks[$i],"_starExp";
106 }
107 for (my $i=0;$i<@marks;$i++) {
108 print LIST "\t",$marks[$i],"_totalExp";
109 }
110 print LIST "\n";
111 print ALN "#>precursor ID \n#precursor sequence\n#precursor structure (mfe)\n#RNA_seq\t@marks\ttotal\n";
112 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";
113 my %moRs;
114
115 foreach my $key (keys %pre) {
116 print ALN ">$key\n$pre{$key}\n$struc{$key}{struc} ($struc{$key}{mfe})\n";
117 next if(! (exists $pre_read{$key}));
118 my @array=@{$pre_read{$key}};
119 @array=sort{$a->[3]<=> $b->[3]} @array;
120
121 my $length=length($pre{$key});
122
123 my $maxline=-1;my $max=0; ### storage the maxinum express read line
124 my $totalReadsNo=0;
125 my @not_over=(); ### new read format better for moRs analysis
126
127 ####print out Aln file start
128 for (my $i=0;$i<@array;$i++) {
129 my $maps=$array[$i][3]+1;
130 my $mape=$array[$i][3]+length($array[$i][4]);
131 my $str="";
132 $str .= "." x ($maps-1);
133 $str .=$array[$i][4];
134 $str .="." x ($length-$mape);
135 $str .=" ";
136
137 $array[$i][0]=~/:([\d|_]+)_x(\d+)$/;
138 my @sample=split /\_/,$1;
139 my $total=$2;
140 print ALN $str,"@sample","\t",$total,"\n";
141
142 if($total>$max){$max=$total; $maxline=$i;}
143 $totalReadsNo+=$total;
144
145 push @not_over,[$key,$maps,$mape,$array[$i][0],$total,"+"];
146 }
147 ####print out Aln file end
148
149 #### express list start
150 my ($ms,$me,$ss,$se);
151 if (!(exists($pre_mature{$key}))) {
152 $ms=$array[$maxline][3]+1;
153 $me=$array[$maxline][3]+length($array[$maxline][4]);
154 ($ss,$se)=&other_pair($ms,$me,$struc{$key}{'struc'});
155
156 my ($mexp,$sexp,$texp)=&express($ms-$upstream,$me+$downstream,$ss-$upstream,$se+$downstream,\@array);
157 print LIST "$key\t$key\tmature:$ms..$me\tstar:$ss..$se\t@$mexp\t@$sexp\t@$texp\n";
158 }
159 else{
160 foreach my $maID (keys %{$pre_mature{$key}}) {
161 $ms=$pre_mature{$key}{$maID}{"mature"}[0];
162 $me=$pre_mature{$key}{$maID}{"mature"}[1];
163 $ss=$pre_mature{$key}{$maID}{"star"}[0];
164 $se=$pre_mature{$key}{$maID}{"star"}[1];
165 my ($mexp,$sexp,$texp)=&express($ms-$upstream,$me+$downstream,$ss-$upstream,$se+$downstream,\@array);
166 print LIST "$maID\t$key\tmature:$ms..$me\tstar:$ss..$se\t@$mexp\t@$sexp\t@$texp\n";
167 }
168 }
169 #### express list end
170
171 #### analysis moRs start
172 my @result; my @m_texp;my $m_texp=0; ### moRs informations
173
174 while (@not_over>0) {
175 my @over=@not_over;
176 @not_over=();
177
178 #丰度最高tag
179 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;
180 for (my $i=0;$i<@over;$i++) {
181 my @m_array=@{$over[$i]};
182 if ($m_max<$m_array[4]) {
183 $m_max=$m_array[4];
184 $m_maxline=$i;
185 }
186 }
187 $m_start=$over[$m_maxline][1];
188 $m_end=$over[$m_maxline][2];
189 $m_exp=$m_max;
190 $over[$m_maxline][3]=~/:([\d|_]+)_x(\d+)$/;
191 my @m_nums=split/_/,$1;
192 for (my $j=0;$j<@m_nums;$j++) {
193 $m_exp[$j]=$m_nums[$j];
194 }
195
196 #统计以丰度最高tag为坐标的reads, 两端位置差异不超过3nt
197 for (my $i=0;$i<@over;$i++) {
198 next if($i==$m_maxline);
199 my @m_array=@{$over[$i]};
200 if (abs($m_array[1]-$m_start)<=3 && abs($m_array[2]-$m_end)<=3) {
201 $m_exp+=$m_array[4];
202 $m_no++;
203 $m_array[3]=~/:([\d|_]+)_x(\d+)$/;
204 my @m_nums=split/_/,$1;
205 for (my $j=0;$j<@m_nums;$j++) {
206 $m_exp[$j] +=$m_nums[$j];
207 }
208 }
209 elsif($m_array[1]>=$m_end || $m_array[2]<=$m_start){push @not_over,[@{$over[$i]}];} #去除跨越block的reads
210 }
211 if($m_exp>5){### 5个reads
212 $m_texp+=$m_exp;
213 for (my $j=0;$j<@m_exp;$j++) {
214 $m_texp[$j]+=$m_exp[$j];
215 }
216 my $string=&subseq($pre{$key},$m_start,$m_end,"+");
217 push @result,"\t$m_start\t$m_end\t@m_exp\t$m_exp\t$m_no\t$string" ;
218 }
219 }
220
221 my $str=scalar @result;
222 my $percent=sprintf("%.2f",$m_texp/$totalReadsNo);
223 $str=">$key\t+\t$m_texp\t$percent\t".$str."\t$pre{$key}";
224 @{$moRs{$str}}=@result;
225
226 #### analysis moRs end
227 }
228
229 ##### moRs print out start
230 foreach my $key (keys %moRs) {
231 my @tmp=split/\t/,$key;
232 next if ($tmp[4]<=2);
233 next if($tmp[3]<0.95);
234 my @over;
235 for (my $i=0;$i<@{$moRs{$key}};$i++) {
236 my @arrayi=split/\t/,$moRs{$key}[$i];
237 for (my $j=0;$j<@{$moRs{$key}};$j++) {
238 next if($i==$j);
239 my @arrayj=split/\t/,$moRs{$key}[$j];
240 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))) {
241 push @over,$moRs{$key}[$i];
242 }
243 }
244 }
245 if (@over>0) {
246 print MORS "$key\n";
247 foreach (@{$moRs{$key}}) {
248 print MORS "$_\n";
249 }
250 }
251 }
252 ###### moRs print out end
253 close ALN;
254 close LIST;
255 close MORS;
256
257 $"=" ";##### reset
258
259
260 ################### Sub programs #################
261 sub express{
262 my ($ms,$me,$ss,$se,$read)=@_;
263 my (@mexp,@sexp,@texp);
264 $$read[0][0]=~/:([_|\d]+)_x(\d+)$/;
265 my @numsample=split/_/,$1;
266 for (my $i=0;$i<@numsample;$i++) {
267 $mexp[$i]=0;
268 $sexp[$i]=0;
269 $texp[$i]=0;
270 }
271
272 for (my $i=0;$i<@{$read};$i++) {
273 my $start=$$read[$i][3]+1;
274 my $end=$$read[$i][3]+length($$read[$i][4]);
275 $$read[$i][0]=~/:([_|\d]+)_x(\d+)$/;
276 my $expresses=$1;
277 my @nums=split/_/,$expresses;
278
279 for (my $j=0;$j<@nums;$j++) {
280 $texp[$j]+=$nums[$j];
281 }
282 if ($start>=$ms && $end<=$me) {
283 for (my $j=0;$j<@nums;$j++) {
284 $mexp[$j]+=$nums[$j];
285 }
286 }
287 if ($start>=$ss && $end<=$se) {
288 for (my $j=0;$j<@nums;$j++) {
289 $sexp[$j]+=$nums[$j];
290 }
291 }
292 }
293 return(\@mexp,\@sexp,\@texp);
294 }
295
296 sub structure{
297 foreach my $key (keys %pre_mature) {
298 if (!(defined $pre{$key})){die "!!!!! No precursor sequence $key, please check it!\n";}
299 #my ($str,$mfe)=RNA::fold($pre{$key});
300 my $rnafold=`perl -e 'print "$pre{$key}"' | RNAfold --noPS`;
301 my @rnafolds=split/\s+/,$rnafold;
302 my $str=$rnafolds[1];
303 my $mfe=$rnafolds[-1];
304 $mfe=~s/\(//;
305 $mfe=~s/\)//;
306
307 $struc{$key}{"struc"}=$str;
308 #$struc{$key}{"mfe"}=sprintf ("%.2f",$mfe);
309 $struc{$key}{"mfe"}=$mfe;
310
311 foreach my $id (keys %{$pre_mature{$key}}) {
312 ($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);
313 }
314 =cut
315 ##### Nucleotide complementary
316 my @tmp=split//,$str;
317 my %a2b;
318 my @bps;
319 for (my $i=0;$i<@tmp;$i++) {
320 if ($tmp[$i] eq "("){push @bps,$i+1 ; next;}
321 if ($tmp[$i] eq ")") {
322 my $up=pop @bps;
323 $a2b{$i+1}=$up;
324 $a2b{$up}=$i+1;
325 }
326 }
327
328 ##### search star position
329 foreach my $id (keys %{$pre_mature{$key}}) {
330 my $n=0;
331 for (my $i=$pre_mature{$key}{$id}{"mature"}[0];$i<=$pre_mature{$key}{$id}{"mature"}[1] ; $i++) {
332 if (defined $a2b{$i}) {
333 my $a=$i; my $b=$a2b{$i};
334 if($a>$b){
335 $pre_mature{$key}{$id}{"star"}[0]=$b-$n+2;
336 $pre_mature{$key}{$id}{"star"}[1]=$b-$n+2+($pre_mature{$key}{$id}{"mature"}[1]-$pre_mature{$key}{$id}{"mature"}[0]);
337 }
338 if($a<$b{
339 $pre_mature{$key}{$id}{"star"}[1]=$b+$n+2;
340 $pre_mature{$key}{$id}{"star"}[0]=$b+$n+2-($pre_mature{$key}{$id}{"mature"}[1]-$pre_mature{$key}{$id}{"mature"}[0]);
341 }
342 last;
343 }
344 $n++;
345 }
346 }
347 =cut
348 }
349 }
350 sub other_pair{
351 my ($start,$end,$structure)=@_;
352 ##### Nucleotide complementary
353 my @tmp=split//,$structure;
354 my %a2b; my @bps;
355 for (my $i=0;$i<@tmp;$i++) {
356 if ($tmp[$i] eq "("){push @bps,$i+1 ; next;}
357 if ($tmp[$i] eq ")") {
358 my $up=pop @bps;
359 $a2b{$i+1}=$up;
360 $a2b{$up}=$i+1;
361 }
362 }
363 ##### search star position
364 my $n=0;my $startpos; my $endpos;
365 for (my $i=$start;$i<=$end ; $i++) {
366 if (defined $a2b{$i}) {
367 my $a=$i; my $b=$a2b{$i};
368 # if($a>$b){
369 # $startpos=$b-$n+2;
370 # $endpos=$b-$n+2+($end-$start);
371 # }
372 # if($a<$b){
373 $endpos=$b+$n+2;
374 if($endpos>length($structure)){$endpos=length($structure);}
375 $startpos=$b+$n+2-($end-$start);
376 if($startpos<1){$startpos=1;}
377 # }
378 last;
379 }
380 $n++;
381 }
382 return ($startpos,$endpos);
383 }
384 sub attachPre{
385 open IN, "<$pre_file_name";
386 my $name;
387 while (my $aline=<IN>) {
388 chomp $aline;
389 if ($aline=~/^>(\S+)/) {
390 $name=$1;
391 next;
392 }
393 $pre{$name} .=$aline;
394 }
395 close IN;
396 }
397 sub readPosOnPre{
398 open IN,"<read_mapped.bwt";
399 while (my $aline=<IN>) {
400 chomp $aline;
401 my @tmp=split/\t/,$aline;
402 my $id=lc($tmp[2]);
403 push @{$pre_read{$tmp[2]}},[@tmp];
404 }
405 close IN;
406 }
407 sub maturePosOnPre{
408 open IN,"<mature_mapped.bwt";
409 while (my $aline=<IN>) {
410 chomp $aline;
411 my @tmp=split/\t/,$aline;
412 my $mm=$tmp[0];
413 # $mm=~s/\-3P|\-5P//i;
414 $mm=lc($mm);
415 my $pm=$tmp[2];
416 $pm=lc($pm);
417
418 # next if ($mm ne $pm);### stringent mapping let7a only allowed to map pre-let7a
419 next if($mm!~/$pm/);
420 # print "$tmp[2]\t$tmp[0]\n";
421 # $pre_mature{$tmp[2]}{$tmp[0]}{"mature"}[0]=$tmp[3]-$upstream;
422 # $pre_mature{$tmp[2]}{$tmp[0]}{"mature"}[0]=0 if($pre_mature{$tmp[2]}{$tmp[0]}{"mature"}[0]<0);
423 # $pre_mature{$tmp[2]}{$tmp[0]}{"mature"}[1]=$tmp[3]+length($tmp[4])-1+$downstream;
424 $pre_mature{$tmp[2]}{$tmp[0]}{"mature"}[0]=$tmp[3]+1;
425 $pre_mature{$tmp[2]}{$tmp[0]}{"mature"}[1]=$tmp[3]+length($tmp[4]);
426 }
427 close IN;
428 }
429 sub mapping{
430 my $err;
431 ## build bowtie index
432 #print STDERR "building bowtie index\n";
433 $err = `bowtie-build $pre_file_name miRNA_precursor`;
434
435 ## map mature sequences against precursors
436 #print STDERR "mapping mature sequences against index\n";
437 $err = `bowtie -p $threads -f -v 0 -a --best --strata --norc miRNA_precursor $mature > mature_mapped.bwt 2> run.log`;
438
439 ## map reads against precursors
440 #print STDERR "mapping read sequences against index\n";
441 $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 2> run.log`;
442
443 }
444
445 sub subseq{
446 my $seq=shift;
447 my $beg=shift;
448 my $end=shift;
449 my $strand=shift;
450
451 my $subseq=substr($seq,$beg-1,$end-$beg+1);
452 if ($strand eq "-") {
453 $subseq=revcom($subseq);
454 }
455 return uc $subseq;
456 }
457
458 sub revcom{
459 my $seq=shift;
460 $seq=~tr/ATCGatcg/TAGCtagc/;
461 $seq=reverse $seq;
462 return uc $seq;
463 }
464
465 sub Time{
466 my $time=time();
467 my ($sec,$min,$hour,$day,$month,$year) = (localtime($time))[0,1,2,3,4,5,6];
468 $month++;
469 $year+=1900;
470 if (length($sec) == 1) {$sec = "0"."$sec";}
471 if (length($min) == 1) {$min = "0"."$min";}
472 if (length($hour) == 1) {$hour = "0"."$hour";}
473 if (length($day) == 1) {$day = "0"."$day";}
474 if (length($month) == 1) {$month = "0"."$month";}
475 #print "$year-$month-$day $hour:$min:$sec\n";
476 return("$year-$month-$day-$hour-$min-$sec");
477 }
478
479 sub usage{
480 print <<"USAGE";
481 Version $version
482 Usage:
483 $0 -r -p -m -mis -t -e -f -tag -o -time
484 mandatory parameters:
485 -p precursor.fa miRNA precursor sequences from miRBase # must be absolute path
486 -m mature.fa miRNA sequences from miRBase # must be absolute path
487 -r reads.fa your read sequences #must be absolute path
488
489 -o output directory
490
491 options:
492 -mis [int] number of allowed mismatches when mapping reads to precursors, default 0
493 -t [int] threads number,default 1
494 -e [int] number of nucleotides upstream of the mature sequence to consider, default 2
495 -f [int] number of nucleotides downstream of the mature sequence to consider, default 5
496 -tag [string] sample marks# eg. sampleA;sampleB;sampleC
497 -time sting #make directory time,default is the local time
498 -h help
499 USAGE
500 exit(1);
501 }
502