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comparison extractSClip.pl @ 0:acc8d8bfeb9a

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author jjohnson
date Wed, 08 Feb 2012 16:59:24 -0500
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1 #!/usr/bin/perl -w
2 use strict;
3 #use lib '/home/jwang2/AssembleTest/Detect/nonSJsrc/dev';
4 use Carp;
5 use Getopt::Long;
6 use English;
7 use Pod::Usage;
8 use Data::Dumper;
9 use Bio::DB::Sam;
10 use File::Temp qw/ tempfile tempdir /;
11 use File::Spec;
12 use File::Basename;
13 use Cwd;
14 use List::MoreUtils qw/ uniq /;
15 #custom packages
16 use SCValidator qw($lowqual_cutoff $min_percent_id $min_percent_hq LEFT_CLIP RIGHT_CLIP);
17 use SVUtil qw($use_scratch $work_dir get_input_bam get_work_dir parse_range is_PCR_dup);
18
19 use constant FQ_BASE_NUMBER => 33;
20
21 my $rmdup = 0;
22 my $print_read = 1;
23 $use_scratch = 0;
24 $min_percent_id = 90;
25 my ($work_dir, $out_dir);
26 my $ref_genome;
27 # input/output
28 my ($out_prefix, $range, $input_bam );
29 my $out_suffix = "sclip.txt";
30 my $paired = 1;
31 my ( $help, $man, $version, $usage );
32 my $optionOK = GetOptions(
33 'i|in|input=s' => \$input_bam,
34 'o|out_dir=s' => \$out_dir,
35 'ref_genome=s' => \$ref_genome,
36 'p=s' => \$out_prefix,
37 'scratch!' => \$use_scratch,
38 'paired!' => \$paired,
39 'rmdup!' => \$rmdup,
40 'lq_cutoff=i' => \$lowqual_cutoff,
41 'min_pct_id=i' => \$min_percent_id,
42 'min_pct_hq=i' => \$min_percent_hq,
43 'print_read!' => \$print_read,
44 'r|range=s' => \$range,
45 'h|help|?' => \$help,
46 'man' => \$man,
47 'usage' => \$usage,
48 'v|version' => \$version,
49 );
50
51 pod2usage(-verbose=>2) if($man or $usage);
52 pod2usage(1) if($help or $version );
53
54 my $start_dir = getcwd;
55 if($input_bam) {
56 croak "The bam file you specified does not exist!\n" unless(-e $input_bam);
57 $input_bam = File::Spec->rel2abs($input_bam);
58 }
59 else{
60 croak "You must specify the input bam file or sample name";
61 }
62 croak "You must provide the reference genome in fasta format!" if(!$ref_genome);
63 croak "The reference genome file you speicified does not exist!\n"
64 unless(-e $ref_genome);
65
66 my $input_base = fileparse($input_bam);
67
68 #setup output dir and workind directory
69 $out_dir = getcwd if(!$out_dir);
70 mkdir $out_dir if(!-e $out_dir || ! -d $out_dir);
71 $work_dir = get_work_dir() if($use_scratch);
72 $work_dir = $out_dir if(!$work_dir);
73 $use_scratch = undef if($work_dir eq $out_dir);
74 chdir($work_dir);
75
76 # figure out output prefix
77 $out_prefix = $input_base if(!$out_prefix);
78
79 my $sam = Bio::DB::Sam->new( -bam => $input_bam, -fasta => $ref_genome);
80
81 my $output_file;
82 my $validator = SCValidator->new();
83 if(!$paired) {
84 $validator->remove_validator("strand_validator");
85 }
86 if($range) {
87 my ($chr, $start, $end) = parse_range($range);
88 my $tmp = $chr;
89 $tmp = $tmp . ".$start" if($start);
90 $tmp = $tmp . ".$end" if($end);
91 $output_file = join('.', $out_prefix, $tmp, $out_suffix);
92 $output_file = File::Spec->catfile($out_dir, $output_file);
93
94 my($pcover, $ncover) = extract_range_sclip(
95 -SAM => $sam,
96 -RANGE => $range,
97 -WORK_DIR => $work_dir,
98 -OUTPUT => $output_file,
99 -VALIDATOR => $validator);
100 $output_file = join('.', $out_prefix, $tmp, "cover");
101 $output_file = File::Spec->catfile($out_dir, $output_file);
102 warn "$output_file file exists and it will be replaced!" if(-e $output_file);
103 $rmdup = 0;
104 #$start = 0 unless $start;
105 #my ($tid) = $sam->header->parse_region($chr);
106 #my $chr_len = $sam->header->target_len->[$tid];
107 #$end = $chr_len unless $end;
108
109 #my ($coverage) = $sam->features(-type => 'coverage', -seq_id => $chr,
110 # -start => $start, -end => $end);
111 #my @cc = $coverage->coverage;
112
113 $rmdup = 0;
114 my($fh, $fname) = tempfile( DIR => $work_dir);
115 foreach my $p (keys(%{$pcover})) {
116 my $c = ($rmdup ? scalar(@{$pcover->{$p}}) : $pcover->{$p});
117 print $fh join("\t", $chr, $p, "+", $c, count_coverage($sam, $chr, $p)), "\n";
118 }
119 foreach my $p (keys(%{$ncover})) {
120 my $c = ($rmdup ? scalar(@{$ncover->{$p}}) : $ncover->{$p});
121 print $fh join("\t", $chr, $p, "-", $c , count_coverage($sam, $chr, $p)), "\n";
122 }
123 system("sort -k 2 -n $fname -o $output_file");
124 system("rm $fname");
125 }
126 else{
127 $output_file = join('.', $out_prefix, $out_suffix);
128 $output_file = File::Spec->catfile($out_dir, $output_file);
129 my $header = $sam->header;
130 my $target_names = $header->target_name;
131 $output_file = join('.', $out_prefix, "cover");
132 my $read_file = join('.', $out_prefix, "sclip.txt");
133 $output_file = File::Spec->catfile($out_dir, $output_file);
134 $read_file = File::Spec->catfile($out_dir, $read_file);
135
136 if(-e $output_file) {
137 warn "$output_file file exists and it will be replaced!";
138 system("rm $output_file");
139 }
140 my @t_names = uniq @{$target_names};
141 foreach my $chr (@t_names){
142 my($fh, $fname) = tempfile( DIR => $work_dir);
143 my($pcover, $ncover) = extract_range_sclip(
144 -SAM => $sam,
145 -RANGE =>$chr,
146 -WORK_DIR => $work_dir,
147 -OUTPUT => $read_file,
148 -VALIDATOR => $validator);
149 foreach my $p (keys(%{$pcover})) {
150 my $c = ($rmdup ? scalar(@{$pcover->{$p}}) : $pcover->{$p});
151 print $fh join("\t", $chr, $p, "+", $c, count_coverage($sam, $chr, $p) ), "\n";
152 }
153 foreach my $p (keys(%{$ncover})) {
154 my $c = ($rmdup ? scalar(@{$ncover->{$p}}) : $ncover->{$p});
155 print $fh join("\t", $chr, $p, "-", $c, count_coverage($sam, $chr, $p)), "\n";
156 }
157 system("sort -k 2 -n $fname -o $fname.sorted");
158 system("cat $fname.sorted >> $output_file");
159 system("rm $fname");
160 system("rm $fname.sorted");
161 }
162 }
163 chdir $start_dir;
164 exit(0);
165
166 sub count_coverage {
167 my ($sam, $chr, $pos, $clip) = @_;
168 if($rmdup) {
169 my @pairs;
170 my $seg = $sam->segment(-seq_id => $chr, -start => $pos, -end => $pos);
171 my $n = 0;
172 my $itr = $seg->features(-iterator => 1);
173 while( my $a = $itr->next_seq) {
174 my $sclip_len = 0;
175 if($clip) {
176 my @cigar_array = @{$a->cigar_array};
177 $sclip_len = $cigar_array[0]->[1] if($cigar_array[0]->[0] eq 'S' && $clip == RIGHT_CLIP);
178 $sclip_len = $cigar_array[$#cigar_array]->[1] if($cigar_array[$#cigar_array]->[0] eq 'S' && $clip == RIGHT_CLIP);
179 }
180 next if(@pairs > 0 && is_PCR_dup($a, \@pairs, $sclip_len));
181 $n++;
182 #return $n if( $n > $max_repetitive_cover);
183 if($a->mpos) {
184 push @pairs, [$a->start, $a->end, $a->mate_start, $a->mate_end, $sclip_len];
185 }
186 else {
187 push @pairs, [$a->start, $a->end, 0, 0, $sclip_len];
188 }
189
190 }
191 return $n;
192 }
193 else{
194 my ($c) = $sam->features(-type => 'coverage', -seq_id=> $chr,
195 -start => $pos, -end => $pos);
196 my @c_d = $c->coverage;
197 return $c_d[0];
198 }
199 }
200
201 sub extract_range_sclip {
202 my %arg = @_;
203 my $sam = $arg{-SAM} || croak "missing -SAM";
204 my $range = $arg{-RANGE} || croak "missing -RANGE";
205 my $output_file = $arg{-OUTPUT} || croak "missing -OUTPUT";
206 my $validator = $arg{-VALIDATOR} || croak "missing -VALIDATOR";
207
208 my($fh, $fname) = tempfile( DIR => $work_dir);
209 my (%plus_cover, %neg_cover);
210 $sam->fetch($range,
211 sub {
212 my $a = shift;
213 my $cigar_str = $a->cigar_str;
214 # print STDERR $a->qname, "\t", $cigar_str, "\n";
215 my @cigar_array = @{$a->cigar_array};
216 return if($a->cigar_str !~ m/S/);
217 if($paired && !$a->proper_pair) { #paired but mate is not mapped
218 $validator->remove_validator("strand_validator");
219 }
220 #return if(!$a->proper_pair && $paired);
221 #return if($paired && !$a->mpos);
222 my ($sclip_len, $ort, $pos, $seq, $qual_str, $qual);
223 $qual_str = join( "", (map { chr $_ + FQ_BASE_NUMBER } $a->qscore));
224 if($cigar_array[0]->[0] eq 'S' && $validator->validate($a, LEFT_CLIP) ) {
225 $sclip_len = $cigar_array[0]->[1]; $ort = "-"; $pos = $a->start;
226 $seq = substr($a->query->dna, 0, $sclip_len );
227 $qual = substr($qual_str, 0, $sclip_len);
228
229 my $print = 1;
230 if($rmdup) {
231 if(exists $neg_cover{$pos}) {
232 $print = 0 if(is_PCR_dup($a, $neg_cover{$pos}, $sclip_len));
233 }
234 else {
235 $neg_cover{$pos} = [];
236 }
237 if($print == 1) {
238 if($a->mpos) {
239 push @{$neg_cover{$pos}}, [$a->start, $a->end, $a->mate_start, $a->mate_end, $sclip_len];
240 }
241 else {
242 push @{$neg_cover{$pos}}, [$a->start, $a->end, 0, 0, $sclip_len];
243 }
244 }
245 }
246 else {
247 if(exists $neg_cover{$pos}) {
248 $neg_cover{$pos}++;
249 }
250 else{
251 $neg_cover{$pos} = 1;
252 }
253 }
254 print $fh join("\t", $a->seq_id, $pos, $ort, $a->qname, $seq, $qual), "\n"
255 if($print_read && $print == 1);
256 }
257
258 if($cigar_array[$#cigar_array]->[0] eq 'S' && $validator->validate($a, RIGHT_CLIP)) {
259 $sclip_len = $cigar_array[$#cigar_array]->[1]; $ort = '+'; $pos = $a->end;
260 my $l = length($a->query->dna);
261 $seq = substr($a->query->dna, $l - $sclip_len);
262 $qual = substr($qual_str, $l - $sclip_len);
263
264 my $print = 1;
265 if($rmdup) {
266 if(exists $plus_cover{$pos}) {
267 $print = 0 if(is_PCR_dup($a, $plus_cover{$pos}, $sclip_len));
268 }
269 else {
270 $plus_cover{$pos} = [];
271 }
272 if($print ==1 ) {
273 if($a->mpos) {
274 push @{$plus_cover{$pos}}, [$a->start, $a->end, $a->mate_start, $a->mate_end, $sclip_len];
275 }
276 else {
277 push @{$plus_cover{$pos}}, [$a->start, 0, 0, $a->mate_end, $sclip_len];
278 }
279 }
280 }
281 else {
282 if(exists $plus_cover{$pos}) {
283 $plus_cover{$pos}++;
284 }
285 else{
286 $plus_cover{$pos} = 1;
287 }
288 }
289 $print = is_PCR_dup($a, $plus_cover{$pos}, $sclip_len) if($rmdup);
290 print $fh join("\t", $a->seq_id, $pos, $ort, $a->qname, $seq, $qual), "\n"
291 if($print_read && $print);
292 }
293 if($paired && !$a->proper_pair) { #paired but mate is not mapped, add back
294 $validator->add_validator("strand_validator");
295 }
296 }
297 );
298 if($print_read) {
299 system("sort -k 2 -n $fname -o $fname.sorted");
300 system("cat $fname.sorted >> $output_file");
301 system("rm $fname");
302 system("rm $fname.sorted");
303 }
304 return(\%plus_cover, \%neg_cover);
305 }
306
307
308 =head1 NAME
309
310 extractSClip.pl - extract positions with soft clipped read in bam file.
311
312
313 =head1 VERSION
314
315 This documentation refers to extractSClip.pl version 0.0.1.
316
317
318 =head1 USAGE
319
320 # extract all positions with soft clipped reads in whole genome:
321 ./extractSClip.pl -i sample.bam -g hg18.fa
322 # extract chr1 positions with soft clipped reads
323 ./extractSClip.pl -i sample.bam -g hg18.fa -r chr1
324
325
326 =head1 REQUIRED ARGUMENTS
327
328 -i: Input bam file.
329 --ref_genome: The genome file in fa file, must be the same used to map reads.
330
331
332 =head1 OPTIONS
333
334 -r: The range of positions need to be extracted. Format: chr1 or chr1:500-5000.
335 -o: The output directory, default is current directory.
336 --scratch: use scracth space, default off.
337 --rmdup: remove PCR dumplicate reads, default on, use --normdup to turn it off.
338 --lq_cutoff: low quality cutoff value, default 20.
339 --min_pct_id: minimum percent identify for the aligned high qual part,default 90.
340 --min_pct_hq: minimum percent high quality for soft clipped part, default 80.
341 --print_read: individual soft-clipped read will be printed, default off.
342 -h, --help: The help page.
343 --man: Print the man page.
344 --usage: Print usage information.
345 -v, --version: print version information.
346
347
348 =head1 DESCRIPTION
349
350 This is a program to extract all soft-clipped positions such that for each position
351 a list of requirements need to be satisfied. More specifically, the orientaion of
352 pair-end read should be satisfied, the minimum percent identify of aligned part
353 need to be satisfied, and the minimum percent of hiqh quality soft-clipped part
354 should be satisfied.
355
356
357 =head1 DEPENDENCIES
358
359 The program depend on several packages:
360 1. Bioperl perl module.
361 2. Bio::DB::Sam, version 1.5 or later, it requires samtools lib installed.
362
363 =head1 BUGS AND LIMITATIONS
364
365 There are no known bugs in this module, but the method is limitted to bam file
366 that has soft-clipping cigar string generated.Please report problems to
367 Jianmin Wang (Jianmin.Wang@stjude.org)
368 Patches are welcome.
369
370 =head1 AUTHOR
371
372 Jianmin Wang (Jianmin.Wang@stjude.org)
373
374
375 =head1 LICENCE AND COPYRIGHT
376
377 Copyright (c) 2010 by St. Jude Children's Research Hospital.
378
379 This program is free software: you can redistribute it and/or modify
380 it under the terms of the GNU General Public License as published by
381 the Free Software Foundation, either version 2 of the License, or
382 (at your option) any later version.
383
384 This program is distributed in the hope that it will be useful,
385 but WITHOUT ANY WARRANTY; without even the implied warranty of
386 MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the
387 GNU General Public License for more details.
388
389 You should have received a copy of the GNU General Public License
390 along with this program. If not, see <http://www.gnu.org/licenses/>.