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4
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1 #!/usr/bin/perl
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2
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3 use strict;
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4 use warnings;
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5 use Getopt::Std;
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6 use File::Basename;
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7 $| = 1;
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8
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9 # Grab and set all options
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10 my %OPTIONS = (a => 0, i => "gene_id", m => "intersection-nonempty", s => "no", t => "exon");
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11 getopts('a:cg:i:m:o:r:s:t:', \%OPTIONS);
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12
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13 die qq(
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14 Usage: HTSeq.pl [OPTIONS] Group1=sample1=<SAM/BAM file> [Group1=sample2=<SAM/BAM file> ... Group2=sampleN=<SAM/BAM file> ...]
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15
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16 OPTIONS: -a STR skip all reads with alignment quality lower than the given minimum value (default: $OPTIONS{a})
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17 -c reduce the matrix by removing any feature with no counts
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18 -g STR the features file in the GFF/GTF format
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19 -i STR GFF attribute to be used as feature ID (default: $OPTIONS{i})
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20 -m STR mode to handle reads overlapping more than one feature. Possible values for <mode> are union, intersection-strict and intersection-nonempty (default: $OPTIONS{m})
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21 -o STR output file name for expression matrix
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22 -r STR the name of the output report
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23 -s STR whether the data is from a strand-specific assay (default: $OPTIONS{s})
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24 -t STR feature type (3rd column in GFF file) to be used, all features of other type are ignored (default, suitable for RNA-Seq and Ensembl GTF files: $OPTIONS{t})
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25
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26 ) if(@ARGV == 0);
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27
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28 my $sam_out;
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29 my @counts;
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30 my @features;
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31 my %report;
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32 my @samplenames;
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33 my $current_group;
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34 my @groups;
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35 my @files;
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36 my $groupcount = 0;
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37 my %grouphash;
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38
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39 foreach my $input (@ARGV) {
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40 my ($group, $sample, $input) = split "::", $input;
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41 if(! defined $grouphash{$group}) {
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42 $groupcount++;
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43 $grouphash{$group} = "G${groupcount}:$group";
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44 }
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45 push @groups, $grouphash{$group};
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46 push @samplenames, $sample;
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47 push @files, $input;
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48 }
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49
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50 for(my $index = 0; $index <= $#files; $index++) {
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51 my $input_file = $files[$index];
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52 my $sample = $samplenames[$index];
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53
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54 # run htseq
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55 my @htseq;
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56 my $COMM;
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57 my $file_type = `file $input_file`;
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58 if(grep /text$/, $file_type ) {
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59 $COMM = "htseq-count -q -m $OPTIONS{m} -s $OPTIONS{s} -a $OPTIONS{a} -t $OPTIONS{t} -i $OPTIONS{i} $input_file $OPTIONS{g}";
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60 @htseq = `$COMM`;
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61 } else {
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62 $COMM = "samtools view $input_file | htseq-count -q -m $OPTIONS{m} -s $OPTIONS{s} -a $OPTIONS{a} -t $OPTIONS{t} -i $OPTIONS{i} - $OPTIONS{g}";
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63 @htseq = `$COMM`;
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64 }
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65
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66 my $row = 0;
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67 $report{$sample} = "Command Used: $COMM\n";
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68
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69 for(my $row = 0; $row <= $#htseq; $row++) {
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70 # store the report is an hash
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71 if(grep /^no_feature|^ambiguous|^too_low_aQual|^not_aligned|^alignment_not_unique/, $htseq[$row]) {
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72 $report{$sample} .= $htseq[$row];
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73 } else {
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74 # store the counts in a matrix
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75 chomp $htseq[$row];
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76 my ($feature, $value) = split "\t", $htseq[$row];
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77 $counts[$row][$index] = $value;
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78 if($input_file eq $files[0]) {
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79 push @features, $feature;
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80 }
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81 }
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82 }
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83 }
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84
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85 # print the matrix
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86 open(MATRIX, ">$OPTIONS{o}") || die "Could Not Create Output File $OPTIONS{o}!\n";
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87 print MATRIX "#\t".join("\t", @groups)."\n";
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88 print MATRIX "#Feature\t".join("\t", @samplenames)."\n";
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89 for(my $row = 0; $row <= $#features; $row++) {
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90 if(defined $OPTIONS{c}) {
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91 my $rowsum = 0;
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92 $rowsum += $_ foreach @{ $counts[$row] };
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93 if(!$rowsum) {
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94 next;
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95 }
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96 }
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97 print MATRIX "$features[$row]\t".join("\t", @{ $counts[$row] })."\n";
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98 }
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99 close(MATRIX);
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100
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101 # print the report
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102 open(REPORT, ">$OPTIONS{r}") || die "Could Not Create Output File $OPTIONS{r}!\n";
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103 print REPORT "$groups[$_]:$samplenames[$_]\n$report{$samplenames[$_]}\n" foreach (0..$#samplenames);
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104 close(REPORT);
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105
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106
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107
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