NAME

Tutorial_pipeline01.pl - An example pipeline for the ViennaNGS toolbox

SYNOPSIS

    perl Tutorial_Pipeline01.pl path/to/R/libraries
    Where path/to/R/libraries should point to the directory containing ggplot2

DESCRIPTION

This script is a showcase for using Bio::ViennaNGS components with a real NGS example.

We start from a file containing ENSEMBL annotation information for human protein-coding genes. We are insterested in finding sequence motifs in close proximity to the gene start (50nt upstream, 10nt into the gene) to identify regulatory regions.

PREREQUITES

For running this tutorial on your local machine you will need a recent version of bedtools as well as the following input files (which can be downloaded here):

hg19_highlyexpressed.bed

OPTIONAL: meme.xml

PIPELINE

The first step is to initialize some variables and generate a chromosome_sizes hash.

  my $bed        = 'hg19_highlyexpressed.bed';
  my $name     = (split(/\./,$bed))[0];
  my $upstream = 50;
  my $into     = 10;
  my $outfile  = "$name.ext$upstream\_fromStart_$into\_downstream.bed";
  my $outfile2 = "$name.ext$upstream\_upstream.bed";
  my %sizes = %{fetch_chrom_sizes('hg19')};

Generate a Bio::ViennaNGS::FeatureChain object

The bed file of interest is parsed, a feature array is generated and passed on to Bio::ViennaNGS::FeatureChain, which creates a new Moose Object of type FeatureChain, containing the original bed entries

  my @featurelist = @{parse_bed6($bed)};

Now we create a Bio::ViennaNGS::FeatureChain from the Bed extracted featurelist above

  my $chain = Bio::ViennaNGS::FeatureChain->new('type'=>'original','chain'=>\@featurelist);

Extend the existing chain for motif analysis

The newly created FeatureChain object will now be extended 50nt upstream of the gene start and 10nt into the gene, to retrieve a bed file which contains the putative sequence motifs.

  my $extended_chain = extend_chain(\%sizes,$chain,0,$into,$upstream,0);

Print extended Bio::ViennaNGS::FeatureChain objects to files

Extended chains are now print out to make them available for external tools like bedtools.

  my $out = $extended_chain->print_chain();
  print $Out $out;
  close ($Out);

Summary of so far used methods

fetch_chrom_sizes<as_string>

Returns a chromosome-sizes hash reference for the specified species, e.g. hg19, mm9, mm10, etc.

parse_bed6<as_string>

Reads a bed6 file and returns a feature array.

Bio::ViennaNGS::FeatureChain->new()<as_method>

Generates a new Bio::ViennaNGS::FeatureChain object from a feature array

Bio::ViennaNGS(extend_chain)<as_method>

Extends a Bio::ViennaNGS::FeatureChain object by given constraints

Sequence analysis

 We now generate FASTA files from the extended bed files using bedtools getfasta method.
  my $bedtools = `bedtools getfasta -s -fi hg19_chromchecked.fa -bed $outfile -fo $name.ext$upstream\_fromStart_$into\_downstream.fa`;
 print STDERR "$bedtools\n" if $?;
  $bedtools = `bedtools getfasta -s -fi hg19_chromchecked.fa -bed $outfile2 -fo $name.ext$upstream\_upstream.fa`;
  print STDERR "$bedtools\n" if $?;

To analyze putative sequence motifs in the newly generated Fasta files, we use two approaches. First we analyze the k-mer content with the Bio::ViennaNGS(kmer_enrichment) method for k-mers of length 6 to 8 nt.

  open(IN,"<","$name.ext$upstream\_fromStart_$into\_downstream.fa") || die ("Could not open $name.ext$upstream\_fromStart_$into\_downstream.fa!\n@!\n");

  my @fastaseqs;
  while(<IN>){
    chomp (my $raw = $_);
    next if ($_ =~ /^>/);
    push @fastaseqs, $raw;
}
  close(IN);

  for (6..8){
    my %kmer = %{kmer_enrichment(\@fastaseqs, $_)};
    my $total = sum values %kmer;
    ### Print Output
    open(KMER,">","$_\_mers") or die "Could not open file $_\_mers$!\n";
    print KMER "$_\-mer\tCount\tRatio\n";
    print KMER "TOTAL\t$total\t1\n";
    foreach my $key  (sort {$kmer{$b} <=> $kmer{$a} } keys %kmer) {
      my $ratio = nearest(.0001,$kmer{$key}/$total);
      print KMER "$key\t$kmer{$key}\t$ratio\n";
    }
    close(KMER);
  }

MEME

In a second approach we run MEME to retrieve the 20 most over-represented motifs of length 8. This can either be done using the MEME command line tool or web-service, or for convenience by simply downloading the output meme.xml file from here

  meme hg19_highexpressed.ext50_fromStart_10_downstream.fa -oc MEME_hg19_highexpressed.ext50_fromStart_10_downstream.fa -w 8 -dna -nmotifs 20

Once the meme run is done, we want to have a nice figure which shows the e-value and site coverage of the top 10 motifs

  my $cmd = "perl ../scripts/MEME_xml_motif_extractor.pl -f Example_Pipeline_meme.xml -r $RLIBPATH -t Example_Pipeline";
  my( $success, $error_message, $full_buf, $stdout_buf, $stderr_buf ) = run(command => $cmd, verbose => 0);

  if(!$success){    
    my $this_function = (caller(0))[3];
    print STDERR "ERROR: MEME_xml_motif_extractor.pl run unsuccessful\n";
    print join "", @$full_buf;
    unless ($r) {
      warn "If you do not provide a valid R-lib-path and ggplot is not found in the standard R path, this pipeline will not be able to parse the MEME xml output.\n";
      pod2usage(-verbose => 0);
    }
  }

AUTHOR

Joerg Fallmann <joerg.fallmann@univie.ac.at>

cpanm Bio::ViennaNGS

perl -MCPAN -e shell
install Bio::ViennaNGS