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| Q1: What are mature miRBase miRNAs not detected by miRDeep2 in the result files? | ||
| A: Those miRNAs have not been detected by miRDeep2 due to unusual precursor structures or too short/long mature sequence lengths. | ||
| The miRDeep2 module does de novo miRNA prediction and miRNA precursors or mature sequences that do not fit the assumed biogenesis | ||
| model were not reported as such. The assignment of being a known miRNA happens in the very end by simple sequence comparison | ||
| with all predicted miRNA precursors. | ||
| However, running the quantification module reports expression values for all known miRNAs in your data | ||
| and thus they will still appear in the miRDeep2 output files in this special category. | ||
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| Q2: How are miRNA expression values reported by the qunatification module normalized? | ||
| A: Expression values are normalized by library size and multiplied by a factor of 10E6 which corresponds | ||
| technically to counts per million mapped miRNA reads. | ||
| The library size is the total number of reads mapping to miRNA precursors. | ||
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| Q3: Does miRDeep2 differential miRNA expression profiling? | ||
| A: No, but the output from the quantification module can be used | ||
| directly as input for specialized differential expression | ||
| profiling tools. | ||
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| #!/usr/bin/perl | ||
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| ####################################################################### | ||
| # | ||
| # | ||
| # This script extracts the mirdeep2 precursors from the result.csv file | ||
| # and writes a fasta file and a bed file for these | ||
| # See 'Usage' for more information. | ||
| # | ||
| # Bed files are only created for the precursor sequences. | ||
| # | ||
| # S.M. Jan. 2012 | ||
| # | ||
| ###################################### | ||
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| use strict; | ||
| use Getopt::Std; | ||
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| my %options=(); | ||
| getopts("r:s:dpht:mkT:o:",\%options); | ||
| if(not -f $options{'r'} or $options{'h'}){ | ||
| die " | ||
| Usage: get_mirdeep2_precursors.pl -r result.csv [options] | ||
| [options] | ||
| -s [int]\toutput only precursors with min-score >= [int] | ||
| -t [int]\toutput only precursors with score < [int] | ||
| -d \toutput dna instead of rna | ||
| -p \tmake simple id with the name only | ||
| -h \tprint this help message | ||
| -m \tget_mature instead of precursor | ||
| -k \tget_star instead of precursor | ||
| -T \tTrackname for bedfiles | ||
| -o \toutdir | ||
| "; | ||
| } | ||
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| my $od="result_mirnas"; | ||
| $od=$options{'o'} if($options{'o'}); | ||
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| if(not -d $od){mkdir $od;} | ||
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| if($options{'T'} !~ /\S+/){ | ||
| $options{'T'} = 'notTrackname'; | ||
| } | ||
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| my @l1=split(/result/,$options{'r'}); | ||
| my ($timestamp,$dmp)=split(".csv",$l1[1]); | ||
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| my ($known,$novel, $not, $line,$thres,$score,$strandcol,$bedh1,$pcoord); | ||
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| my $bedh="browser position BPOS | ||
| browser hide all | ||
| track name=\"TNAME\" description=\"TDESC\" visibility=2 | ||
| itemRgb=\"On\"; | ||
| "; | ||
| my $first=1; | ||
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| my @line; | ||
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| $thres = -50; | ||
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| if(defined $options{'s'}){ $thres = $options{'s'};} | ||
| $score=$thres; | ||
| my $max='na'; | ||
| my $maxs=999999999999999999999999999; | ||
| if(defined $options{'t'}){ | ||
| $max=$options{'t'}; | ||
| $maxs=$max; | ||
| } | ||
| open IN,"$options{'r'}" or die "No results.csv file given\n"; | ||
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| my $seqcol=15; | ||
| $seqcol=13 if($options{'m'}); | ||
| $seqcol=14 if($options{'k'}); | ||
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| my @names=qw(0 1 2 3 4 5 6 7 8 9 10 11 12 mature star pres); | ||
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| while(<IN>){ | ||
| if(/novel miRNAs predicted by miRDeep2/){ | ||
| $novel=1; | ||
| $known=0; | ||
| $not=0; | ||
| $line=<IN>; | ||
| $first=1; | ||
| open OUT,">$od/novel_$names[$seqcol]${timestamp}_score${score}_to_$max.fa"; | ||
| open BED,">$od/novel_$names[$seqcol]${timestamp}_score${score}_to_$max.bed"; | ||
| next; | ||
| }elsif(/miRBase miRNAs detected/){ | ||
| close OUT; | ||
| close BED; | ||
| $novel=0; | ||
| $known=1; | ||
| $not=0; | ||
| $line=<IN>; | ||
| $first=1; | ||
| close OUT; | ||
| close BED; | ||
| open OUT,">$od/known_$names[$seqcol]${timestamp}_score${score}_to_$max.fa"; | ||
| open BED,">$od/known_$names[$seqcol]${timestamp}_score${score}_to_$max.bed"; | ||
| next; | ||
| }elsif(/miRBase miRNAs not detected by miRDeep2/){ | ||
| $novel=0; | ||
| $known=0; | ||
| $not=1; | ||
| $first=1; | ||
| close OUT; | ||
| close BED; | ||
| open OUT,">$od/not_$names[$seqcol]${timestamp}_score${score}_to_$max.fa"; | ||
| open BED,">$od/not_$names[$seqcol]${timestamp}_score${score}_to_$max.bed"; | ||
| next; | ||
| }else{} | ||
| next if(/^\s*$/); | ||
| if($novel or $known){ | ||
| chomp; | ||
| @line=split(/\t/); | ||
| my $coord='na'; | ||
| $coord=$line[16] if($line[16]); | ||
| if($known){ | ||
| if($line[1] >= $thres and $line[1] < $maxs){ | ||
| if($options{'d'}){ | ||
| $line[$seqcol] =~ tr/uU/tT/; | ||
| } | ||
| if($options{'p'}){ | ||
| if($line[0] =~ /\|([a-zA-Z0-9_-]*)$/){ | ||
| $line[0] = $1; | ||
| } | ||
| print OUT ">$line[0]\n",uc($line[$seqcol]),"\n"; | ||
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| }else{ | ||
| print OUT ">${line[0]}_${line[9]}_x${line[5]}_coord:",$coord,"_score:$line[1]\n",uc($line[$seqcol]),"\n"; | ||
| } | ||
| chomp $coord; | ||
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| $strandcol="0,0,255"; | ||
| if($coord =~ /^(\S+):(\d+)\.\.(\d+):(\S)$/){ | ||
| $pcoord="$1:$2-$3"; | ||
| $strandcol="255,0,0" if($4 eq "+"); | ||
| if($first){ | ||
| $first=0; | ||
| $bedh1=$bedh; | ||
| $bedh1 =~ s/TNAME/$options{'T'}.known_miRNAs/; | ||
| $bedh1 =~ s/TDESC/known miRNAs detected by miRDeep2 for $options{'T'}/; | ||
| $bedh1 =~ s/BPOS/$pcoord/; | ||
| print BED "$bedh1"; | ||
| } | ||
| print BED "$1\t$2\t$3\t$line[0]\t$line[1]\t$4\t$2\t$3\t$strandcol\n"; | ||
| } | ||
| } | ||
| }else{ | ||
| if($line[1] >= $thres and $line[1] < $maxs){ | ||
| if($options{'d'}){ | ||
| $line[$seqcol] =~ tr/uU/tT/; | ||
| } | ||
| if($options{'p'}){ | ||
| if($line[0] =~ /\|([a-zA-Z0-9_-]*)$/){ | ||
| $line[0] = $1; | ||
| } | ||
| print OUT ">$line[0]\n",uc($line[$seqcol]),"\n"; | ||
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| }else{ | ||
| print OUT ">${line[0]}_x${line[5]}_coord:",$coord,"_score:$line[1]\n",uc($line[$seqcol]),"\n"; | ||
| } | ||
| chomp $coord; | ||
| $strandcol="0,0,255"; | ||
| if($coord =~ /^(\S+):(\d+)\.\.(\d+):(\S)$/){ | ||
| $strandcol="255,0,0" if($4 eq "+"); | ||
| if($first){ | ||
| $first=0; | ||
| $bedh1=$bedh; | ||
| $bedh1 =~ s/TNAME/$options{'T'}.novel_miRNAs/; | ||
| $bedh1 =~ s/TDESC/novel miRNAs detected by miRDeep2 for $options{'T'}/; | ||
| $bedh1 =~ s/BPOS/$pcoord/; | ||
| print BED "$bedh1"; | ||
| } | ||
| print BED "$1\t$2\t$3\t$line[0]\t$line[1]\t$4\t$2\t$3\t$strandcol\n"; | ||
| } | ||
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| } | ||
| next; | ||
| } | ||
| } | ||
| if($not){ | ||
| chomp; | ||
| @line=split(/\t/); | ||
| my $coord='na'; | ||
| $coord=$line[16] if($line[16]); | ||
| if($options{'d'}){ | ||
| $line[$seqcol] =~ tr/uU/tT/; | ||
| } | ||
| if($options{'p'}){ | ||
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| }else{ | ||
| print OUT ">${line[0]}_x${line[4]}\n",uc($line[$seqcol]),"\n"; | ||
| } | ||
| chomp $coord; | ||
| $strandcol="0,0,255"; | ||
| if($coord =~ /^(\S+):(\d+)\.\.(\d+):(\S)$/){ | ||
| $strandcol="255,0,0" if($4 eq "+"); | ||
| if($first){ | ||
| $first=0; | ||
| $bedh1=$bedh; | ||
| $bedh1 =~ s/TNAME/$options{'T'}.not_detected_miRNAs/; | ||
| $bedh1 =~ s/TDESC/miRNAs not detected by miRDeep2 for $options{'T'}/; | ||
| $bedh1 =~ s/BPOS/$pcoord/; | ||
| print BED "$bedh1"; | ||
| } | ||
| print BED "$1\t$2\t$3\t$line[0]\t$line[1]\t$4\t$2\t$3\t$strandcol\n"; | ||
| } | ||
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| next; | ||
| } | ||
| } | ||
| close OUT; |
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