0
|
1 #!/usr/bin/perl -w
|
|
2
|
|
3 ############################################
|
|
4 # tophatstatsPE
|
|
5 # John Garbe
|
|
6 # May 29, 2012
|
|
7 #
|
|
8 # Calculate statistics about the data going into and out of tophat:
|
|
9 # -Count the number of raw reads, the number of aligned reads, the number
|
|
10 # of properly paired reads, etc
|
|
11 #
|
|
12 ###########################################
|
|
13
|
|
14 die "USAGE: tophatstats.pl accepted_hits.bam fastqfile.fastq\n" unless ($#ARGV == 1);
|
|
15
|
|
16 $bamfile = $ARGV[0];
|
|
17 $fastqfile = $ARGV[1];
|
|
18 die "cannot open $fastqfile" unless (-e $fastqfile);
|
|
19 die "cannot open $bamfile" unless (-e $bamfile);
|
|
20
|
|
21 my $junk;
|
|
22 $divide = 4; # four lines per read in a fastq file
|
|
23 $insertsum = 0;
|
|
24 $insertcount = 0;
|
|
25 # count total number of raw reads
|
|
26 if (1) {
|
|
27 print "Input files: $bamfile\t $fastqfile\n";
|
|
28 $results = `wc -l $fastqfile`;
|
|
29 ($results, $junk) = split ' ', $results;
|
|
30 $total = $results / $divide;
|
|
31 print "$total total read pairs in fastq file\n";
|
|
32 }
|
|
33
|
|
34 # read in bam file as a pipe from samtools
|
|
35 open IFILE, "samtools view $bamfile |" or die "cannot open $bamfile\n";
|
|
36
|
|
37 # initialize array counting multiple alignments
|
|
38 for $i (0..200) {
|
|
39 $count[$i] = 0;
|
|
40 for $j (0..40) {
|
|
41 $unique[$i][$j] = 0;
|
|
42 }
|
|
43 }
|
|
44
|
|
45 $insertsum = 0;
|
|
46 $insertcount = 0;
|
|
47 # read through bam file, counting alignments
|
|
48 while ($line = <IFILE>) {
|
|
49 @line = split /\t/, $line, 3;
|
|
50 $count[$line[1]]++;
|
|
51
|
|
52 # pull out number of alignments for this read
|
|
53 @line = split /\t/, $line, 12;
|
|
54 ($junk, $int) = split /NH:i:/, $line[11];
|
|
55 ($int, $junk) = split /\W/, $int;
|
|
56 if ($int == 1) { # count unique alignments
|
|
57 $unique[$line[1]][1]++;
|
|
58 } elsif ($int > 1) { # count non-unique alignments
|
|
59 $unique[$line[1]][2]+= 1 / $int;
|
|
60 }
|
|
61 if ($line[1] == 99 || $line[1] == 83) { # if proper insert size use for insert size average
|
|
62 @line = split /\t/, $line, 10;
|
|
63 $insertsum += $line[8];
|
|
64 $insertcount++;
|
|
65 }
|
|
66 }
|
|
67
|
|
68 # singleton
|
|
69 $singleton_unique = 0;
|
|
70 $singleton_multiple = 0;
|
|
71 for $i (73, 133, 89, 121, 165, 181, 101, 117, 153, 185, 69, 137) {
|
|
72 $singleton_unique += $unique[$i][1];
|
|
73 $singleton_multiple += int($unique[$i][2]);
|
|
74 }
|
|
75 $singleton = $singleton_unique + $singleton_multiple;
|
|
76 $singleton_percent = $singleton / $total * 100;
|
|
77 printf "$singleton (%.2f%%) read pairs with only one read in the pair mapped ($singleton_unique with unique alignments)\n", $singleton_percent;
|
|
78
|
|
79 # correct correct
|
|
80 $correctcorrect_unique = 0;
|
|
81 $correctcorrect_multiple = 0;
|
|
82 for $i (99, 83) {
|
|
83 $correctcorrect_unique += $unique[$i][1];
|
|
84 $correctcorrect_multiple += int($unique[$i][2]);
|
|
85 }
|
|
86 $correctcorrect = $correctcorrect_unique + $correctcorrect_multiple;
|
|
87 $correctcorrect_percent = $correctcorrect / $total * 100;
|
|
88 printf "$correctcorrect (%.2f%%) read pairs mapped with correct orientation and insert size ($correctcorrect_unique with unique alignments)\n", $correctcorrect_percent;
|
|
89
|
|
90 # correct wrong insert size
|
|
91 $correctwrong_unique = 0;
|
|
92 $correctwrong_multiple = 0;
|
|
93 for $i (81, 97, 65, 113) {
|
|
94 $correctwrong_unique += $unique[$i][1];
|
|
95 $correctwrong_multiple += int($unique[$i][2]);
|
|
96 }
|
|
97 $correctwrong = $correctwrong_unique + $correctwrong_multiple;
|
|
98 $correctwrong_percent = $correctwrong / $total * 100;
|
|
99 printf "$correctwrong (%.2f%%) read pairs mapped with correct orientation but wrong insert size ($correctwrong_unique with unique alignments)\n", $correctwrong_percent;
|
|
100
|
|
101 # correct wrong orientation
|
|
102 $correctswitched_unique = 0;
|
|
103 $correctswitched_multiple = 0;
|
|
104 for $i (67, 115) {
|
|
105 $correctswitched_unique += $unique[$i][1];
|
|
106 $correctswitched_multiple += $unique[$i][2];
|
|
107 }
|
|
108 $correctswitched = $correctswitched_unique + $correctswitched_multiple;
|
|
109 $correctswitched_percent = $correctswitched / $total * 100;
|
|
110 printf "$correctswitched (%.2f%%) read pairs mapped with wrong orientation but correct insert size ($correctswitched_unique with unique alignments)\n", $correctswitched_percent;
|
|
111
|
|
112 # no mapping
|
|
113 $nomapping = $total - ($correctcorrect + $correctswitched + $correctwrong + $singleton);
|
|
114 $nomapping_percent = $nomapping / $total * 100;
|
|
115 printf "$nomapping (%.2f%%) read pairs with no mapping\n", $nomapping_percent;
|
|
116
|
|
117 # insert size
|
|
118 $insertavg = 0;
|
|
119 $insertavg = $insertsum / $insertcount unless ($insertcount == 0);
|
|
120 #printf "%.2fbp average inner distance between read pairs (of read pairs with correct insert size)\n", $insertavg; # Not ready for primetime yet - needs verification
|
|
121
|
|
122 exit;
|
|
123
|
|
124 ############################################################
|
|
125
|