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PileupLong.pm
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package Pileup;
#################################
#Dan MacLean Dec 30th 2009 [email protected]
#module of methods for dealing with alignments
#################################
use strict;
use Data::Dumper;
use Exporter;
use Storable qw(nstore retrieve);
use Encode;
use Bio::SeqIO;
use Solexa::Bowtie;
use vars qw($VERSION @ISA @EXPORT @EXPORT_OK %EXPORT_TAGS);
$VERSION = 0.1;
@ISA = qw(Exporter);
@EXPORT = ();
@EXPORT_OK = qw();
%EXPORT_TAGS = (DEFAULT => [qw()],
ALL =>[qw()]);
sub new {
my $class = shift;
my %arg;
###very messily go through the args array and set up an easier hash.....
for (my $i= 0; $i< scalar(@_); ++$i){
if ($_[$i] eq '-format'){
$arg{'-format'} = $_[$i+1];
}
if ($_[$i] eq '-file'){
$arg{'-file'} = $_[$i+1];
}
if ($_[$i] eq '-refs'){
$arg{'-refs'} = $_[$i+1];
}
}
die "unknown alignment format, allowed are bowtie bwt maq bin binary \n\n" unless $arg{'-format'} =~ m/bowtie|bwt|maq|binary|bin/i;
die "no alignment file provided or not recognised\n\n" unless $arg{'-file'};
my $self = {};
if ($arg{'-format'} =~ m/bowtie|bwt/){
die "no reference file provided, required for bowtie style alignments\n\n" unless $arg{'-refs'};
$$self{'refs_file'} = $arg{'-refs'};
}
$$self{'format'} = $arg{'-format'};
$$self{'file'} = $arg{'-file'};
if ($$self{'format'} =~ /maq/ ){
open FILE, "<$$self{'file'}" || die "Can't open maq file $$self{'file'} \n\n";
warn "Building a map for a maq file $$self{'file'}, please wait\n\n";
while (my $line = <FILE>){
my @tmp = split(/\s+/, $line);
$$self{'_length'}{$tmp[0]} = $tmp[1];
#next if exists $$self{'_index'}{$tmp[0]};
my $start = tell(FILE);
$line = encode('UTF-8', $line);
$$self{'_index'}{$tmp[0]} = $start - length($line); ##works through the pileup file and records the distance in bytes into the file that each contig starts
$$self{'_point_index'}{$tmp[0]}{$tmp[1]} = $start - length($line); ## list of individual reference in the file
$$self{'_ref_base'}{$tmp[0]}{$tmp[1]} = $tmp[2];
$$self{'_nucleotides_aligned'} = ($$self{'_nucleotides_aligned'}) + $tmp[3]; #warn $tmp[3], "\n";
$$self{'_contig_nucs_aligned'}{$tmp[0]} += $tmp[3];
$$self{'_length'}{$tmp[0]} = $tmp[1];
$$self{'_depth'}{$tmp[0]}{$tmp[1]} = $tmp[3];
}
close FILE;
}
elsif ($$self{'format'} =~ /bowtie|bwt/ ){
die "Can't open reference sequence file $$self{'refs_file'} \n\n" unless -e $$self{'refs_file'};
open FILE, "<$$self{'file'}" || die "Can't open bowtie file $$self{'file'}\n\n";
"warn loading reference sequences into memory\n\n";
my $infile = Bio::SeqIO->new(-file => "$$self{'refs_file'}", -format => 'fasta');
while (my $seq = $infile->next_seq){
$$self{'_references'}{$seq->id} = $seq->seq;
$$self{'_length'}{$seq->id} = $seq->length;
}
warn "Building a map for a bowtie file, please wait\n\n";
while (my $line = <FILE>){
my @tmp = split(/\s+/, $line);
my $start = tell(FILE);
$line = encode('UTF-8', $line);
push @{$$self{'_index'}{$tmp[2]}}, ($start - length($line)); ##works through the bowtie file and records the distance in bytes into the file that each match to each contig starts
for (my $i = $tmp[3]; $i <= $tmp[3] + length($tmp[4]); $i++){
$$self{'_nucleotides_aligned'}++;
$$self{'_contig_nucs_aligned'}{$tmp[2]}++;
$$self{'_depth'}{$tmp[2]}{$i}++;
$$self{'_ref_base'}{$tmp[2]}{$i} = substr($$self{'_references'}{$tmp[2]}, $i, 1) unless defined $$self{'_ref_base'}{$tmp[2]}{$i};
}
}
close FILE;
}
elsif ($$self{'format'} =~ /binary|bin/ or -B $$self{'file'}){
$self = retrieve_pickle($arg{'-file'});
return $self;
}
unless (-e "$arg{'-file'}.binary"){
warn "writing binary version of file to \n\n";
$self = bless($self, $class);
$self->pickle("$arg{'-file'}.binary");
}
return bless($self, $class);
}
sub format{
my $self = shift;
return $$self{'format'};
}
sub file{
my $self= shift;
return $$self{'file'};
}
sub refs_file{
my $self = shift;
return $$self
}
#sub _new_maq {
#my $class = shift;
#my $file = shift || die;
#my $self = {};
#warn $file, "\n";
#$$self{'_file'} = $file;
# open FILE, "<$file" || die "Can't open $file\n\n";
# while (my $line = <FILE>){
#chomp $line;
# my @tmp = split(/\s+/, $line);
# next if exists $$self{'_index'}{$tmp[0]};
# my $start = tell(FILE);
# $line = encode('UTF-8', $line);
# $$self{'_index'}{$tmp[0]} = $start - length($line); ##works through the pileup file and records the distance in bytes into the file that each contig starts
# warn "$tmp[0]\n";
# }
# close FILE;
# warn "Index built for $file\n\n";
# return $self;
# bless $self, $class;
#}
sub _get_pile{
my $self = shift;
my $contig = shift;
if ($self->format =~ /maq/){
$self->_get_pile_maq($contig);
}
elsif($self->format =~ /bowtie|bwt/){
$self->_get_pile_bowtie($contig);
}
return $self;
}
sub _get_pile_bowtie{
my $self = shift;
my $contig = shift;
my %pileup;
my %stack;
### get the reference sequence and populate the reference hash
die "cant find reference sequence ...\n\n" unless exists $$self{'_references'}{$contig};
for (my $i = 1; $i<=length($$self{'_references'}{$contig}); $i++){
$pileup{$contig}{$i}{'_ref_base'} = substr($$self{'_references'}{$contig}, $i - 1, 1);
$pileup{$contig}{$i}{'_coverage_depth'} = 0;
$stack{$contig}{$i} = '@'
}
##bowtie is indexed from 0!!
foreach my $seekto (@{$$self{'_index'}{$contig}}){
my $file = $self->file;
open FILE, "<$file";
seek(FILE, $seekto, 0);
my $line = <FILE>;
my $bwt = new Bowtie($line);
die "mismatched contig name\n\n" unless $contig eq $bwt->targetname;
my %mm = $bwt->mismatches;
for (my $i = ($bwt->alignstart) + 1; $i <= $bwt->alignstop; $i++){
##increment the read coverage
$pileup{$contig}{$i}{'_coverage_depth'}++;
##make a maq like stack ...
##add the mismatches if any to the stack
my $ref = $self->get_ref_base($contig, $i);
if (exists $mm{$i - 1}){
if ($bwt->strand eq '+'){
#if (defined $stack{$contig}{$i} ){
$stack{$contig}{$i} = $stack{$contig}{$i} . uc($mm{$i - 1}{$ref});
# }
#else{
# $stack{$contig}{$i} = '@';
#}
}
else{
#if (defined $stack{$contig}{$i}){
#my %conv = { 'A' => 'T'. "T" => "A", "G" => "C", "C" => "G"};
$stack{$contig}{$i} = $stack{$contig}{$i} . lc($mm{$i - 1}{$ref});
#}
#else {
# $stack{$contig}{$i} = '@';
#}
}
}
## add the matches to the stack
else {
if ($bwt->strand eq '+'){
#if (defined $stack{$contig}{$i}){
$stack{$contig}{$i} = $stack{$contig}{$i} . '.';
#}
#else {
# $stack{$contig}{$i} = '@';
#}
}
else {
#if (defined $stack{$contig}{$i}){
$stack{$contig}{$i} = $stack{$contig}{$i} . ',';
#}
#else {
# $stack{$contig}{$i} = '@';
#}
}
}
}
}
##make the pileup information from the new made stack
for (my $i = 1; $i<=length($$self{'_references'}{$contig}); $i++){
my $ref = $self->get_ref_base($contig,$i);
my $st = $stack{$contig}{$i};
#warn $ref, "\t", $st, "\t", $i, "\n";
my @al = split(/|/, $st);
shift @al;
#### NB strand information is lost here...!
foreach my $b (@al){
if($b =~ m/\./){
$pileup{$contig}{$i}{$ref}++;
}
elsif ($b =~ m/,/){
$pileup{$contig}{$i}{$ref}++;
}
elsif ($b =~ m/n/i){
$pileup{$contig}{$i}{'N'}++;
}
elsif ($b eq 'A' or $b eq 'C' or $b eq 'G' or $b eq 'T' ){
$pileup{$contig}{$i}{$b}++;
}
else {
my $u = uc($b);
#my $n = $comp{$u};
$pileup{$contig}{$i}{$u}++;
}
}
}
$$self{'_pileup'}= \%pileup;
$$self{'_stack'}= \%stack;
return $self;
}
sub _get_pile_maq{
my $self = shift;
my $contig = shift;
# warn "get pile called, getting pile for $contig\t";
my %pileup;
my %stack;
my $seekto = $$self{'_index'}{$contig};
open FILE, "<$$self{'file'}";
seek(FILE, $seekto, 0);
while (my $line = <FILE>){
chomp $line;
my @tmp = split(/\s+/, $line);
last unless $contig eq $tmp[0];
$pileup{$tmp[0]}{$tmp[1]}{'_ref_base'} = $tmp[2];
$pileup{$tmp[0]}{$tmp[1]}{'_coverage_depth'} = $tmp[3];
$stack{$tmp[0]}{$tmp[1]} = $tmp[4];
my @bases = qw(A C G T N );
foreach my $b (@bases){
$pileup{$tmp[0]}{$tmp[1]}{$b} = 0;
}
my %comp = ('A' => 'T', 'G' => 'C', 'T' => 'A', 'C' => 'G' );
my @al = split(/|/, $tmp[4]);
shift @al;
#### NB strand information is lost here...!
foreach my $b (@al){
if($b =~ m/\./){
$pileup{$tmp[0]}{$tmp[1]}{$tmp[2]}++;
}
elsif ($b =~ m/,/){
$pileup{$tmp[0]}{$tmp[1]}{$tmp[2]}++;
}
elsif ($b =~ m/n/i){
$pileup{$tmp[0]}{$tmp[1]}{'N'}++;
}
elsif ($b eq 'A' or $b eq 'C' or $b eq 'G' or $b eq 'T' ){
$pileup{$tmp[0]}{$tmp[1]}{$b}++;
}
else {
my $u = uc($b);
#my $n = $comp{$u};
$pileup{$tmp[0]}{$tmp[1]}{$u}++;
}
}
}
close FILE;
$$self{'_pileup'}= \%pileup;
$$self{'_stack'} = \%stack;
return $self;
}
sub stack{
my $self = shift;
my $contig = shift;
my $pos = shift;
return $$self{'_stack'}{$contig}{$pos};
}
sub name_file{
my $self = shift;
return $self->file;
}
sub make_binary{
my $self = shift;
my $filename = shift;
$self->pickle($filename);
}
sub load_binary{
my $file = shift;
my $pileup = retrieve_pickle($file);
return $pileup;
}
sub pickle {
my $self = shift;
my $filename = shift;
nstore($self, $filename);
return;
}
sub retrieve_pickle{
my $file = shift;
my $pileup = Storable::retrieve("$file");
return $pileup;
}
sub get_coverage{ ## gets coverage for a single position on a particular contig
my $self = shift;
my $contig = shift;
my $pos = shift;
return $$self{'_depth'}{$contig}{$pos};
}
sub get_ref_base{
my $self = shift;
my $cont = shift;
my $pos = shift;
if ($self->format =~ /maq/){
return $$self{'_ref_base'}{$cont}{$pos};
}
elsif ($self->format =~ /bwt|bowtie/){
return substr($$self{'_references'}{$cont}, $pos - 1, 1);
}
}
sub get_contigs{ ## returns list of contigs
my $self = shift;
#my $size = keys %{$$self;
return keys %{$$self{'_index'}};
}
sub contig_count{ ## returns number of contigs
my $self = shift;
if ($self->format =~ /maq|bwa/){
my $size = keys %{$$self{'_index'}};
return $size;
}
elsif ($self->format =~ /bowtie|bwt/){
my $size = keys %{$$self{'_references'}};
return $size;
}
}
sub contig_length{ ## returns a contig length
my $self = shift;
my $cont = shift;
if (!defined $cont){
warn "Undefined contig";
return 0;
}
if (!exists $$self{'_index'}{$cont}){
warn "contig not in set ..!";
return 0;
}
return $$self{'_length'}{$cont};
#$self->_get_pile($cont) unless exists $$self{'_pileup'}{$cont};
#my @unsorted = keys %{$$self{'_pileup'}{$cont}};
#my @sorted = sort {$b <=> $a } @unsorted;
#return shift @sorted;
}
sub nt_coverage{ ## returns hash of number of each nt called at a position
my $self = shift;
my $cont = shift;
my $pos = shift;
if (!defined $pos or !defined $cont){
warn "Undefined position";
return;
}
if (!exists $$self{'_index'}{$cont}){
warn "contig not in set ..!";
return 0;
}
$self->_get_pile($cont) unless exists $$self{'_pileup'}{$cont};
my %t = %{$$self{'_pileup'}{$cont}{$pos}};
return %t;
}
sub get_consensus{
my $self = shift;
my $contig = shift;
my $position = shift;
my %cov = $self->nt_coverage($contig,$position);
my $max = 0;
my $consensus = 'none';
foreach my $nt (keys %cov){
if ($cov{$nt} > $max){
$consensus = $nt;
}
}
foreach my $nt (keys %cov){
if ($cov{$nt} == $max){
$consensus = $consensus .'/'. $nt;
}
}
return $consensus;
}
sub average_depth{ ## returns average depth of coverage over all contigs
my $self = shift;
# my $num = 0;
# if (exists $$self{'_average_depth'}){
#return $$self{'_average_depth'};
# }
# else{
# my $total = $self->total_nucleotides();
# my @contigs = $self->get_contigs();
# foreach my $c (@contigs){
# $num += $self->contig_average_depth($c);
# }
# my $average_depth = $num / scalar(@contigs);
# $$self{'_average_depth'} = $average_depth;
# return $average_depth;
# }#
my $total_depth = 0;
my @contigs = $self->get_contigs();
while (my $contig = shift @contigs){
$total_depth += $self->contig_length($contig);
}
return ($$self{'_nucleotides_aligned'} / $total_depth);
}
sub contig_average_depth{ ## returns single contig average depth
my $self = shift;
my $contig = shift;
if (!defined $contig){
warn "Undefined contig";
return 0;
}
return $self->nucleotides_aligned_to_contig($contig) / $self->contig_length($contig);
}
sub total_nucleotides{ ## returns number of nucleotides in all contigs ie sum of lengths
my $self = shift;
my $length = 0;
if (exists $$self{'_total_nucleotides'}){
return $$self{'_total_nucleotides'};
}
else {
my @contigs = $self->get_contigs;
foreach my $c (@contigs) {
$length += $self->contig_length($c);
}
$$self{'_total_nucleotides'} = $length;
return $length;
}
}
sub nucleotides_aligned_to_contig{ ## returns the sum of aligned nucleotides at each position in a contig
my $self = shift;
my $contig = shift;
if (!defined $contig){
warn "Undefined contig";
return;
}
return $$self{'_contig_nucs_aligned'}{$contig};
}
sub nucleotides_aligned{ ## returns the sum of nucleotides at each position over all contigs
my $self = shift;
my $num = 0;
return $$self{'_nucleotides_aligned'};
}
sub _make_pileup_array { ### makes a 2D array of the maq pileup with the reference in the first column
my $self = shift;
my $contig = shift;
if (!exists $$self{'_index'}{$contig}){
warn "contig not in set ..!";
return 0;
}
$self->_get_pile($contig) unless exists $$self{'_pileup'}{$contig};
my @pile;
my $max_cov = 0;
for (my $r = 1; $r<= $self->contig_length($contig); $r++){
$max_cov = $self->get_coverage($contig,$r) if $self->get_coverage($contig,$r) > $max_cov;
my $ref = $self->get_ref_base($contig,$r);
my $st = $self->stack($contig, $r);
my @al = split(/|/, $st);
#shift @al;
$pile[$r - 1 ][0] = $ref;
for (my $i = 0; $i <= $max_cov; ++$i){
my $read_base = $al[$i];
next unless defined $read_base;
if ($read_base eq '.'){
$pile[$r-1][$i] = $ref;
}
elsif ($read_base eq ','){
$pile[$r-1][$i] = lc($ref);
}
elsif($read_base =~ m/[ATCG]/i) {
$pile[$r-1][$i] = $read_base;
}
}
}
###now fill out all the blank spaces in the array ...
for (my $i = 0; $i < $self->contig_length($contig); $i++){
for (my $j = 0; $j < $max_cov; $j++){
$pile[$i][$j] = '.' unless defined $pile[$i][$j];
}
}
return \@pile;
}
sub _rotate_array { ##makes a rotation of a 2D array ..
my $self = shift;
my $array = shift;##ref to array to rotate
my @rotated_array;
#foreach my
}
sub html_pile { ## converts a 2d array in a pretty html format
my $self = shift;
my $contig = shift;
my $orient = shift;
$orient = 'up' unless defined $orient;
my $pile = $self-> _make_pileup_array($contig);
#my $rotated = $self->_rotate_array(\$pile);
my $length = scalar(@{$$pile[0]});# get the length of the inner array
my @html;
if ($orient eq 'up'){
for (my $i = $length; $i>= 0; $i--){
for (my $j = 0; $j < scalar(@$pile); $j++){
my $h = '';
my $el = $$pile[$j][$i];
my $ref = $$pile[$j][0];
my $p = $j + 1;
if ($i == 0){
$h = "<span title=\"$p\" class='ref'>" . $el . '</span>';
}
else {
if (uc($el) =~ m/^$ref$/){
if ($el =~ m/A/i){
$h = "<span title=\"$p\" class='A'>" . $el . '</span>';
}
elsif ($el =~ m/T/i){
$h = "<span title=\"$p\" class='T'>" . $el . '</span>';
}
elsif ($el =~ m/G/i){
$h = "<span title=\"$p\" class='G'>" . $el . '</span>';
}
elsif ($el =~ m/C/i){
$h = "<span title=\"$p\" class='C'>" . $el . '</span>';
}
else{
$h = '<span class=\'X\'>' . ' ' . '</span>';
}
}
else{
if ($el =~ m/A/i){
$h = "<span title=\"$p\" class='different_A'>" . $el . '</span>';
}
elsif ($el =~ m/T/i){
$h = "<span title=\"$p\" class='different_T'>" . $el . '</span>';
}
elsif ($el =~ m/G/i){
$h = "<span title=\"$p\" class='different_G'>" . $el . '</span>';
}
elsif ($el =~ m/C/i){
$h = "<span title=\"$p\" class='different_C'>" . $el . '</span>';
}
else{
$h = "<span title=\"$p\" class=\'X\'>" . ' ' . '</span>';
}
}
}
push @html, $h if defined $h;
}
push @html, '<br>',"\n";
}
}
elsif ($orient eq 'down'){
for (my $i = 0; $i <= $length; $i++){
for (my $j = 0; $j < scalar(@$pile); $j++){
my $h = '';
my $el = $$pile[$j][$i];
my $ref = $$pile[$j][0];
my $p = $j + 1;
if ($i == 0){
$h = "<span title=\"$j\" class='ref'>" . $el . '</span>';
}
else{
if (uc($el) =~ m/$ref/){
if ($el =~ m/A/i){
$h = "<span title=\"$p\" class='A'>" . $el . '</span>';
}
elsif ($el =~ m/T/i){
$h = "<span title=\"$p\" class='T'>" . $el . '</span>';
}
elsif ($el =~ m/G/i){
$h = "<span title=\"$p\" class='G'>" . $el . '</span>';
}
elsif ($el =~ m/C/i){
$h = "<span title=\"$p\" class='C'>" . $el . '</span>';
}
else{
$h = '<span title=\"$p\" class=\'X\'>' . ' ' . '</span>';
}
}
else{
if ($el =~ m/A/i){
$h = "<span title=\"$p\" class='different_A'>" . $el . '</span>';
}
elsif ($el =~ m/T/i){
$h = "<span title=\"$p\" class='different_T'>" . $el . '</span>';
}
elsif ($el =~ m/G/i){
$h = "<span title=\"$p\" class='different_G'>" . $el . '</span>';
}
elsif ($el =~ m/C/i){
$h = "<span title=\"$p\" class='different_C'>" . $el . '</span>';
}
else{
$h = "<span title=\"$p\" class=\'X\'>" . ' ' . '</span>';
}
}
}
push @html, $h if defined $h;
}
push @html, '<br>',"\n";
}
}
return join '', @html;
#return $rotated;
}
sub html_style{
my $sheet = $_[1];
if ($sheet eq 'block'){
return '
<!--
body {
background-color: black;
font-family: Andale mono, monospace;
font-size: 14px;
color: black;
}
.A {
background-color: #00FFFF;
}
.C {
background-color: #FFFF00;
}
.T {
background-color: #00FF00;
}
.G {
background-color: #FF0000;
}
.ref {
background-color: white;
}
-->
';
}
elsif ($sheet eq 'simple'){
return '
<!--
body {
background-color: black;
font-family: Andale mono, monospace;
font-size: 14px;
color: white;
}
.A {
color: #00FFFF;
}
.C {
color: #FFFF00;
}
.T {
color: #00FF00;
}
.G {
color: #FF0000;
}
.ref {
color: white;
}
-->
';
}
elsif ($sheet eq 'difference'){
return '<!--
body {
background-color: black;
font-family: Andale mono, monospace;
font-size: 14px;
color: black;
}
.A {
color: #00FFFF;
}
.C {
color: #FFFF00;
}
.T {
color: #00FF00;
}
.G {
color: #FF0000;
}
.different_A{
background-color: #00FFFF;
}
.different_C{
background-color: #FFFF00;
}
.different_T {
background-color: #00FF00;
}
.different_G {
background-color: #FF0000;
}
.ref{
background-color: white;
}
-->';
}
}
1;
=head1 NAME
Pileup - module that creates object representing maq format pileup files. Will soon include methods for converting bowtie and SOAP
alignments into the pileup format for easy later analysis.
=head1 AUTHOR
Dan MacLean ([email protected])
=head1 SYNOPSIS
use Solexa::Pileup;
my $pileup = new Pileup(/home/macleand/Desktop/mypile);
=head1 DESCRIPTION
Pileup is the text based generic, easy to read output from the maq series of alignment programs. The format allows simple representation of
alignments, describing only the depth of coverage at each position on a reference sequence, the reference sequence itself and the
and the identities of nucleotides that align over that position. It does not record anything to do with quality scores or anything to do
with the actual reads that make up the alignment, like identity or mate pairs.
=head1 PREREQUISITES