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unique.pl
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unique.pl
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#!/usr/bin/perl
use warnings;
use strict;
use Getopt::Std;
my $program = 'unique'; #name of script
my $seq_recog = "acgtn\?:~-"; # MOST AMBIGUITY CODES NOT RECOGNISED FOR MOST SEQUENCES
my @changes = qw(A->T A->C A->G C->A C->G C->T G->A G->T G->C T->A T->C T->G);
my @bases = qw(A C G T);
my %parameters; #input and output file names
my ($datafile, $prefix,$outfile,$fastaindex,$lengthfile,$nonuniquefile,$insertionfile,$ambancfile,$hapfile,$hapseqfile);
my (@names, %seq);
my $alignment_length = 0;
my $refpattern = 'ORF';
my $insthreshold = 0;
my $getuniqueatshared = "no";
my $ucscpos = "no";
my $includeref = "no";
my $hotspotlength = 7;
my (@refseqnames, @neighbour_changes, @neighbour_bases);
($prefix = $program) =~ s/(.*?).pl/$1/; # default output prefix
getopts('i:n:m:o:r:I:H:fhtsCNcuRMk',\%parameters);
if (exists($parameters{"i"})) { $datafile = $parameters{"i"}; }
if (exists($parameters{"n"})) { $datafile = $parameters{"n"}; }
if (exists($parameters{"m"})) { $datafile = $parameters{"m"}; }
if (exists($parameters{"o"})) { $prefix = $parameters{"o"}; }
if (exists($parameters{"r"})) { $refpattern = $parameters{"r"}; }
if (exists($parameters{"R"})) { $includeref = "yes"; }
if (exists($parameters{"I"})) {
if (defined $parameters{"I"}) { $insthreshold = $parameters{"I"}; }
} # OPTIONAL:
if (exists($parameters{"u"})) { $ucscpos = "yes"; } #default is to output as 1..100
if (exists $parameters{"f"}) { $fastaindex = $prefix."_withindex.fa"; } #default fastaindex filename
if (exists $parameters{"s"}) { $nonuniquefile = $prefix."_nonunique.fa"; } #default nonunique filename
if (exists $parameters{"N"}) { $getuniqueatshared = "yes"; } #count unique point subs at shared sites (manual labour required by user for final calls)
if (exists $parameters{"H"}) { $hotspotlength = $parameters{"H"}; }
if (exists $parameters{'c'}) {
foreach my $change (@changes) { foreach (0,1,2,"?") { push (@neighbour_changes, "$_$change"); } }
unless (exists $parameters{"N"}) { foreach (0,1,2,"?") { push (@neighbour_changes, "$_?"); } }
foreach my $base (@bases) { foreach (0,1,2,"?") { push (@neighbour_bases, "$_$base"); } }
}
# NOT OPTIONAL:
$outfile = $prefix.".subs"; #default outfile name
$lengthfile = $prefix.".lengths"; #default length filename
$insertionfile = $prefix.".ins"; #default insertionfile name
$ambancfile = $prefix."ambanc.xls"; #default ambiguous ancestors filename
$hapfile = $prefix.".haps"; #default haplotype filename
$hapseqfile = $prefix.".haps.gfa"; #default haplotype alignment filename
print "\n\t< $program > - Counting unique subs in an alignment\n\n";
unless ((exists $parameters{"i"}) || (exists $parameters{"n"}) || (exists $parameters{"m"})) {
print "\n USAGE: $program -i <datafile>\n";
print " OR: $program -n <datafile>\n";
print " OR: $program -m <datafile>\n\n";
print " -i\tdatafile: sequence alignment in fasta format\n";
print " -n\tdatafile: sequence alignment in nexus format\n";
print " -m\tdatafile: sequence alignment in mase format\n";
print " -o\tprefix for output files [default: $prefix]\n";
print " -r\tpattern for reference sequence names [default: 1st sequence]\n";
print " \tto define open reading frame(s).\n";
print " -k\tcreate a majority consensus to use as reference sequence\n";
print " \tto define open reading frame(s) or direction of indels.\n";
print " -R\tinclude reference sequence(s) when deciding unique sites\n";
print " \tunique changes in reference sequences are not counted.\n";
print " -M\tmerge identical sequences into a single haplotype\n";
print " -t\tdo not count 5' truncation as deletions [e.g. for nonLTRs].\n";
print " -C\ttreat complex events as separate insertions and deletions.\n";
print " -N\tcount unique point subs at non-unique sites.\n";
print " -H\tminimum length of homopolymer runs in reference seq to report [$hotspotlength bp]\n";
print " -c\tshow nearest neighbour context for unique point subs.\n";
print " -I\tminimum length of Insertions to print to [prefix]_ins.fa\n";
print " -s\tprint sites with non-unique point subs to fasta file [prefix]_nonunique.fa.\n";
print " -f\tprint fasta alignment with index as 1st seq.\n";
print " -u\treport nucleotide positions in ucsc format ie 0 based start, 1 based end [1..100].\n";
print " -h\thide headers for output tables.\n\n";
print "NOTE: this script does not recognize ambiguity codes e.g. RYWS etc. Convert these to Ns before using. fastaLC2n.pl with the -A option will do this. Also this script needs too much memory for genome alignments. Use al2variable.pl to reduce the size of your alignment to variable sites only in that case.\n\n";
exit;
}
print "\ncommand: $program ";
foreach my $flag (sort keys %parameters) {
print "-$flag ";
if (defined $parameters{$flag}) { print "$parameters{$flag} "; }
}
print "\n\n";
## READ IN SEQUENCES FROM FILE
#
my $seq_ref;
if (exists $parameters{'n'}) { ($seq_ref,$alignment_length,@names) = nexus($datafile); } # NEXUS FORMAT
elsif (exists $parameters{'m'}) { ($seq_ref,$alignment_length,@names) = mase($datafile); } # MASE FORMAT
else { ($seq_ref,$alignment_length,@names) = fasta($datafile); } # FASTA FORMAT
%seq = %$seq_ref;
# TRANSLATE TO UPPERCASE TO AVOID PATTERN MATCHING ISSUES LATER AND GET RID OF UNUSUAL CHARACTERS
#
print "\nChecking case. ";
my ($case, $converted);
foreach my $name (@names) {
for (my $i=0; $i < @{$seq{$name}}; $i++) {
if ($seq{$name}[$i] =~ /[acgt]/) { $seq{$name}[$i] =~ tr/acgt/ACGT/; $case++; }
if ($seq{$name}[$i] !~ /[ACGT?-]/) {
unless (defined $converted) {
$converted = "Converted $seq{$name}[$i] to ?.";
}
$seq{$name}[$i] = "?";
}
}
}
if (defined $case) { print "Converted lower-case bases to upper-case.\n"; }
else { print "Case is unchanged.\n"; }
if (defined $converted) { print "$converted\n"; }
## MERGE IDENTICAL SEQUENCES INTO A SINGLE HAPLOTYPE
# # Note: Ref is undetermined when deciding haplotypes
if (exists $parameters{'M'}) {
print "\n";
my %seenhap;
my @newnames;
open HAPS, ">$hapfile" or die "couldn't open $hapfile : $!";
open HAPSEQ, ">$hapseqfile" or die "couldn't open $hapseqfile : $!";
foreach my $name1 (@names) { # foreach seq
if ($seenhap{$name1}) { next; }
my @hapnames;
foreach my $name2 (@names) { # identify all matching seqs
if ($name1 eq $name2) { next; }
my $diff = "no";
for (my $i=0; $i < @{$seq{$name1}}; $i++) {
if ($seq{$name1}[$i] eq $seq{$name2}[$i]) { next; }
elsif (($seq{$name1}[$i] eq "?") || ($seq{$name2}[$i] eq "?")) { next; }
elsif ($seq{$name1}[$i] ne $seq{$name2}[$i]) { $diff = "yes"; last; }
}
if ($diff eq "no") {
push(@hapnames, $name2);
$seenhap{$name2}++;
}
}
my $minamb = $alignment_length; # identify which seq has the fewest ambiguous sites
my $hapname;
foreach my $name ($name1, @hapnames) {
my $amb = 0;
foreach (@{$seq{$name}}) { if ($_ eq "?") { $amb++; } }
if ($amb < $minamb) { $hapname = $name; $minamb = $amb; }
}
my @others;
foreach (@hapnames) { if ($hapname ne "$_") { push (@others, $_); } }
print "$hapname: @others\n";
print HAPS "$hapname: @others\n";
push(@newnames, $hapname);
}
print "\nOriginal seqnames: @names\nNew merged hapnames: @newnames\n";
print HAPS "\nOriginal seqnames: @names\nNew merged hapnames: @newnames\n";
@names = @newnames;
}
## REFERENCE SEQUENCE(S)
# # $refseqnames are removed from @names index but %seq is kept
if (exists $parameters{'r'}) { # > 1 refseq to be IDed using user provided $refpattern
my @newnames;
foreach (@names) {
if ($_ =~ /$refpattern/i) { push(@refseqnames, $_); }
else { push (@newnames, $_); }
}
@names = @newnames;
}
elsif (exists $parameters{'k'}) { # create a majority consensus to use as reference
push(@refseqnames, "consensus");
for (my $i=0; $i < @{$seq{$names[0]}}; $i++) {
my %seenbase;
foreach my $name (@names) { $seenbase{$seq{$name}[$i]}++; }
if (defined $seenbase{'?'}) { # count ? as non-gaps
foreach my $base (keys %seenbase) {
if (($base eq "?") || ($base eq "-")) { next; }
$seenbase{$base} += $seenbase{'?'};
}
}
my $max = 0;
my $majbase;
foreach my $base (keys %seenbase) { # check through all possibilities at each site in alignment in random order
if ($seenbase{$base} > $max) { $majbase = $base; $max = $seenbase{$base}; }
}
push (@{$seq{"consensus"}},$majbase);
}
}
else { push(@refseqnames, shift @names); } # only 1st seq is treated as refseq
# print out merged haplotypes with majority consensus if these options have been selected
#
if (exists $parameters{'M'}) {
my @printnames = @names;
if (exists $parameters{'k'}) { @printnames = ("consensus", @names); }
foreach (@printnames) {
my $seq = join ("",@{$seq{$_}});
print HAPSEQ ">$_\n$seq\n";
}
close HAPSEQ;
}
## CHECK REFERENCE SEQUENCE(S) FOR HOMOPOLYMER REPEATS
#
my ($previous, $base);
my $start = 0;
my $run = 1;
my $longrun = 0;
foreach my $refname (@refseqnames) {
print "\nChecking for homopolymer runs (>= $hotspotlength) in $refname sequence (possible indel hotspots):\n";
for (my $i=0; $i < $alignment_length; $i++) {
if ($seq{$refname}[$i] =~ /([acgt])/i) {
unless (defined $previous) { $previous = $1; next; }
if ($1 eq $previous) {
if ($start == 0) { $start = $i; } # start of a run
$run++;
$base = $1;
}
else { # end of a run
if ($run >= $hotspotlength) {
print "$datafile $refname homopolymer run ($base)n $run bp long at positions $start .. ".($i-1).".\n";
$longrun++;
}
$previous = $1; # rezero for next run search
$run = 1;
$start = 0;
}
}
}
}
if ($longrun == 0) { print "$datafile @refseqnames has no homopolymer runs.\n"; }
## CREATE CODON POSITION INDEX FROM REFERENCE SEQUENCES
#
my @index;
my (%codon_pos,$started,$finished);
my ($conflicts, @conflicts, %seenconflicting, $conflictstart, $conflictend);
for (my $i=0; $i < $alignment_length; $i++) {
my @with_seq;
foreach my $refname (@refseqnames) {
unless (defined $codon_pos{$refname}) { $codon_pos{$refname} = 0; }
if ($seq{$refname}[$i] =~ /[?a-z]/i) { # ALL AMBIGUITY CODES ARE RECOGNISED FOR REFSEQ
unless (defined $started) { $started = $i; } # INDEX START
$finished = $i;
push(@with_seq,$refname);
$codon_pos{$refname} = codon_incr($codon_pos{$refname});
}
}
unless (defined $started) { push(@index,"8"); } # reference sequence has not started yet
elsif (@with_seq == 0) { push(@index,"-"); }
elsif (@with_seq == 1) { push(@index,$codon_pos{$with_seq[0]}); }
elsif (@with_seq > 1) { # > 1 reference sequence
my $codon_pos;
for (my $j=0; $j < @with_seq; $j++) { # check if there's a conflict for the codon position
if ($j==0) { $codon_pos = $codon_pos{$with_seq[0]}; }
elsif ($codon_pos != $codon_pos{$with_seq[$j]}) {
unless ($seenconflicting{$with_seq[0]}++) { push(@conflicts, $with_seq[0]); }
unless ($seenconflicting{$with_seq[$j]}++) { push(@conflicts, $with_seq[$j]); }
unless (defined $conflictstart) { $conflictstart = $i+1; }
$conflictend = $i+1;
$codon_pos = 0; # call as 0 if there is disagreement on codon position and save details of refs involved
}
}
push(@index,$codon_pos);
if ($codon_pos == 0) { $conflicts++; }
}
}
if (defined $conflicts) { warn "\nReference sequences (@conflicts) show different codon positions at $conflicts positions ($conflictstart..$conflictend) : conflicts will be called as codon position 0.\n"; }
if (@index != $alignment_length) { die "problem making the index (".@index." bp) for the alignment ($alignment_length bp)\n"; }
if ($finished != $alignment_length - 1) { # reference sequence has finished
for (my $i=$finished+1; $i < $alignment_length; $i++) {
$index[$i] = 9;
}
}
# PRINT A FASTA FILE WITH INDEX IF REQUESTED
#
if (exists $parameters{'f'}) {
open INDEXFA, ">$fastaindex" or die "couldn't open $fastaindex : $!";
my $index = join ("",@index);
my $nogaps;
($nogaps = $index) =~ tr/-98//d;
print INDEXFA ">index ".(length $nogaps)." bp\n$index\n";
foreach my $name (@refseqnames,@names) {
my $seq = join("",@{$seq{$name}});
print INDEXFA ">$name\n$seq\n";
}
}
print "\nNo. of reference sequences :\t".@refseqnames."\nReference sequences :\t\t@refseqnames\nNo. of sequences to analyse :\t".@names."\n";
## MAIN
#
open OUT, ">$outfile" or die "couldn't open $outfile : $!";
open LENGTHS, ">$lengthfile" or die "couldn't open $lengthfile : $!";
if (exists $parameters{'I'}) { open INSERTIONS, ">$insertionfile" or die "couldn't open $insertionfile : $!"; }
# HEADER
unless (exists $parameters{'h'}) {
print OUT "name\ttype\tvalue\tAstart .. Aend\tcodon_pos\n";
print LENGTHS "name\tinitial_length\tfinal_length\tinsertion_length\tnon_unique_length\tfewseq_length\talphaI\talphaU\talphaU1st\talphaU2nd\talphaU3rd\talphaU0\talphaUa\talphaUc\talphaUg\talphaUt";
if ($getuniqueatshared eq "yes") {
# print "\talphaU?";
if (exists $parameters{'c'}) {
foreach my $type (@neighbour_changes) { print LENGTHS "\talphaU$type"; }
}
else {
foreach my $change (@changes,"?") { print LENGTHS "\talphaU$change"; }
}
}
elsif (exists $parameters{'c'}) {
foreach my $type (@neighbour_bases) { print LENGTHS "\talphaU$type"; }
}
print LENGTHS "\n";
}
my (%initial_length,%final_length,%insertion_length,%ins_allpos,%del_allpos,%ins_starts,%ins_ends,%ins_length,%start,%del_starts,%del_ends,%del_length,@allins_lengths,@alldel_lengths,@allcomplex_types,@allCdel_lengths,%ins_class,%del_class);
my (%unique_ps,%unique_ps_type,%unique_ps_codon,%shared_pos,%fewseq_length);
my (%ps_length,%alphaU,%alphaI,%alphaIJustin);
my (%complex_types,%complex_starts,%complex_ends,%Cdel_length,%complex_class);
my (%ambanc,%ambancestors);
my $unique_ps_count = 0;
foreach my $name (@names) {
$initial_length{$name} = 0; # zero lengths at start to avoid nulls later
$final_length{$name} = 0;
$insertion_length{$name} = 0;
$fewseq_length{$name} = 0;
$ps_length{$name} = 0;
$alphaU{$name} = 0;
foreach my $codon_position (1,2,3,0) {
$alphaU{"$name**$codon_position"} = 0;
}
foreach my $base ("A", "C", "G", "T", "?") { # do not use qw with "?"
$alphaU{"$name$base"} = 0;
}
foreach my $change (@changes) {
$alphaU{"$name$change"} = 0;
}
if ((exists $parameters{'c'}) && ($getuniqueatshared eq "no")) {
foreach my $type (@neighbour_bases) { $alphaU{"$name$type"} = 0; }
}
elsif (exists $parameters{'c'}) {
foreach my $type (@neighbour_changes) { $alphaU{"$name$type"} = 0; }
}
for (my $i=0; $i < $alignment_length; $i++) {
# INSERTIONS DEFINED BY A "-" IN INDEX WHERE NOT AT UNDEFINED START OR END OF INDEX
#
if (($seq{$name}[$i] =~ /[a-z]/i) && ($index[$i] eq '-')) { # INSERTION
push (@{$ins_allpos{$name}},$i); # store every ins pos within orf region
} # index is 8 or 9 away from orf, never '-'
# DELETIONS DEFINED BY A "-" IN THIS SEQUENCE WHERE INDEX IS NOT "-" AND THIS SEQUENCE HAS STARTED
#
if (!defined $start{$name}) {
unless (exists $parameters{'t'}) { $start{$name} = 0; } # COUNT 5' truncation AS DELS
# DO NOT COUNT 5' truncation AS DELS
elsif ($seq{$name}[$i] =~ /[a-z]/i) { $start{$name} = $i; }
}
# DELETION
if ((defined $start{$name}) && ($index[$i] ne '-') && ($seq{$name}[$i] eq '-')) {
push (@{$del_allpos{$name}},$i); # store every del pos
}
# POINT SUBS
#
if (($seq{$name}[$i] =~ /[a-z]/i) && ($index[$i] =~ /[0123]/)) {
my (%bases,$not_unique,%point_sub, @others);
my $count_other = 0;
my $point_sub = 0;
if ($includeref eq "yes") { @others = (@refseqnames, @names); }
else { @others = @names; }
foreach my $other (@others) {
if (($other ne $name) && ($seq{$other}[$i] =~ /[a-z]/i)) {
$count_other++;
$bases{$seq{$other}[$i]}++; # grab other base states for ancestor
# not unique flag
if ($seq{$other}[$i] eq $seq{$name}[$i]) { $not_unique++; }
else {
$point_sub++; # point sub flag
$point_sub{$seq{$other}[$i]}++; # are all other bases unique?
}
}
}
my %allbases = %bases; # are all sites unique?
$allbases{$seq{$name}[$i]}++; # too little info to infer ancestry?
my $not_all_unique;
if (($count_other + 1) != (keys %allbases)) { $not_all_unique++; }
# NEED AT LEAST 3 SEQS
# THAT ARE NOT ALL UNIQUE
if (($count_other >= 2) && (defined $not_all_unique)) {
my $non_unique_flag;
# NON-UNIQUE POINT SUB
if ((defined $not_unique) && ($point_sub > 1)) {
my $not_all_others_unique;
foreach my $base (keys %point_sub) {
if ($point_sub{$base} > 1) { $not_all_others_unique++; }
# print "hello: $base $not_all_others_unique\n";
}
if (defined $not_all_others_unique) { # do not include site if all other bases
# are unique
$shared_pos{$i}++;
$non_unique_flag++;
}
}
# UNIQUE POINT SUB
elsif (($point_sub > 0) && (!defined $not_unique)) {
my @temp = ancestor(%bases);
my $ancestral = shift @temp;
# print "hello1: $name [$i] $ancestral->$seq{$name}[$i]\n";
push(@{$unique_ps{$name}}, $i);
push(@{$unique_ps_codon{$name}}, $index[$i]);
if (exists $parameters{'c'}) {
my ($nbefore,$nafter) = neighbours(\@{$seq{$name}},$i,\@index);
push(@{$unique_ps_type{$name}}, "$nbefore$ancestral->$seq{$name}[$i]$nafter");
}
else {
push(@{$unique_ps_type{$name}}, "$ancestral->$seq{$name}[$i]");
# if ($ancestral =~ /\[/) { print "ERROR: $name\t$ancestral\t$i\n"; }
}
$unique_ps_count++;
# print OUT "$name\tunique_ps\t$ancestral->$seq{$name}[$i]\t$i .. $i\t$index[$i]\n";
}
# POINT SUB LENGTH COUNTERS (SEQ FOR INDEX, FOR THIS STUDY SEQ AND AT LEAST 2 OTHER SEQS) SHARED DIFFS INCLUDED.
# store lengths in %ps_length: key = nameindex_state length, or key = name for total_length
$ps_length{"$name$index[$i]"}++;
$ps_length{$name}++;
# NUMBER OF AVAILABLE BASES FOR DIFFERENT TYPES OF UNIQUE POINT SUBSTITUTION
#
# GRAB ANCESTRAL STATE
my ($ancestral, @non_uniquebases) = ancestor(%allbases);
# EXCLUDING NON-UNIQUE POINT SUBS
if ($getuniqueatshared eq "no") {
unless (defined $non_unique_flag) {
# do not count this base even if this
# particular seq was unique.
if (@non_uniquebases == 1) {
$alphaU{"$name**$index[$i]"}++; # codon position count
$alphaU{$name}++;
$alphaU{"$name$ancestral"}++;
# AVAILABLE BASES with NEAREST NEIGHBOUR
if (exists $parameters{'c'}) {
my @neighbours = neighbours(\@{$seq{$name}},$i,\@index);
my $at = atsummary(@neighbours);
$alphaU{"$name$at$ancestral"}++;
}
}
else {
$shared_pos{$i}++;
}
}
}
else { # NOT EXCLUDING NON-UNIQUE POINT SUBS
my $seq = $seq{$name}[$i];
$alphaU{$name}++;
# adjusted codon position count
$alphaU{"$name**$index[$i]"} += (4 - @non_uniquebases)/3;
# ONLY ONE NON-UNIQUE BASE.
# ANCESTOR UNAMBIGUOUS
if (@non_uniquebases == 1) {
$alphaU{"$name$ancestral"}++;
foreach my $base (@bases) {
if ($base =~ $ancestral) { next; } # skip because not a change
# possible changes resolved
# AVAILABLE BASES with NEAREST NEIGHBOUR
if (exists $parameters{'c'}) {
my @neighbours = neighbours(\@{$seq{$name}},$i,\@index);
my $at = atsummary(@neighbours);
$alphaU{"$name$at$ancestral->$base"}++;
}
# AVAILABLE BASES
else { $alphaU{"$name$ancestral->$base"}++; }
}
}
else { # > 1 NON-UNIQUE BASE
if (defined $non_unique_flag) { # but base has not changed uniquely so do
# not need to infer ancestor
$alphaU{"$name$seq"}++;
foreach my $base (@bases) {
# could have been ancestral so
if ($base =~ /$ancestral/) { next; } # sub type not available
# possible changes resolved
# AVAILABLE BASES with NEAREST NEIGHBOUR
if (exists $parameters{'c'}) {
my @neighbours = neighbours(\@{$seq{$name}},$i,\@index);
my $at = atsummary(@neighbours);
$alphaU{"$name$at$seq->$base"}++;
}
# AVAILABLE BASES
else { $alphaU{"$name$seq->$base"}++; }
}
}
else { # ANCESTOR AMBIGUOUS
$alphaU{"$name?"}++;
if (exists $parameters{'c'}) {
my @neighbours = neighbours(\@{$seq{$name}},$i,\@index);
my $at = atsummary(@neighbours);
$alphaU{"$name$at?"}++;
}
# gather data for sites that have
# unique subs with ambiguous ancestors
# for print (to assist manual phylogenetic)
# resolution
push(@{$ambanc{$i}}, $name);
$ambancestors{$i} = $ancestral;
}
}
}
}
else { $fewseq_length{$name}++; }
}
# LENGTHS. NEED COUNTERS TO DEAL WITH END EFFECTS OF DELETIONS THAT EXTEND BEYOND REFERENCE SEQUENCE.
# (length does not include extensions)
if ((defined $start{$name}) # deals with truncation status (see DELETION)
&& ($seq{$name}[$i] =~ /[a-z-]/i) && ($index[$i] =~ /[0123]/)) {
$initial_length{$name}++; # size at time of arrival
# before deletions or insertions
}
if ((defined $start{$name})
&& ($seq{$name}[$i] =~ /[a-z]/i) && ($index[$i] =~ /[0123-]/)) {
$final_length{$name}++; # size after deletions and insertions
}
if ((defined $start{$name})
&& ($seq{$name}[$i] =~ /[a-z]/i) && ($index[$i] =~ /-/)) {
$insertion_length{$name}++;
}
}
# SUMMARISE SUBSTITUTIONS
#
# SUMMARISE POINT SUBS
if ($getuniqueatshared eq "no") { # do not count unique ps at non-unique sites
if (defined @{$unique_ps{$name}}) {
my (@new_unique_ps,@new_unique_ps_type,@new_unique_ps_codon);
for (my $i=0; $i < @{$unique_ps{$name}}; $i++) {
if (exists $shared_pos{$unique_ps{$name}[$i]}) {
#$alphaU{"$name$index[$unique_ps{$name}[$i]]"}--;
#$alphaU{$name}--;
$unique_ps_count--;
}
else {
push (@new_unique_ps, $unique_ps{$name}[$i]);
push (@new_unique_ps_type, $unique_ps_type{$name}[$i]);
push (@new_unique_ps_codon, $unique_ps_codon{$name}[$i]);
}
}
@{$unique_ps{$name}} = @new_unique_ps;
@{$unique_ps_type{$name}} = @new_unique_ps_type;
@{$unique_ps_codon{$name}} = @new_unique_ps_codon;
}
}
# SUMMARISE INSERTIONS
my ($ins_startsref,$ins_endsref,$ins_lengthref) = starts_and_length(\@{$ins_allpos{$name}},\@index,\@{$seq{$name}});
@{$ins_starts{$name}} = @$ins_startsref;
@{$ins_ends{$name}} = @$ins_endsref;
@{$ins_length{$name}} = @$ins_lengthref;
# SUMMARISE DELETIONS
my ($del_startsref,$del_endsref,$del_lengthref) = starts_and_length(\@{$del_allpos{$name}},\@index,,\@{$seq{$name}});
@{$del_starts{$name}} = @$del_startsref;
@{$del_ends{$name}} = @$del_endsref;
@{$del_length{$name}} = @$del_lengthref;
# SUMMARISE COMPLEX EVENTS
unless (exists $parameters{'C'}) {
my (%remove_ins,@new_del_starts,@new_del_ends,@new_del_length,@new_ins_starts,@new_ins_ends,@new_ins_length);
for (my $j = 0; $j < @{$del_starts{$name}}; $j++) {
my $complex_event_flag;
my $previous_pos = $del_starts{$name}[$j] - 1;
my $next_pos = $del_ends{$name}[$j] + 1;
for (my $k = 0; $k < @{$ins_ends{$name}}; $k++) {
if ($ins_ends{$name}[$k] == $previous_pos) { # insertion then deletion?
# COMPLEX EVENT
# store complex event characters
push (@{$complex_types{$name}}, "+$ins_length{$name}[$k]-$del_length{$name}[$j]");
push (@{$complex_starts{$name}}, $ins_starts{$name}[$k]);
push (@{$complex_ends{$name}}, $del_ends{$name}[$j]);
push (@{$Cdel_length{$name}}, $del_length{$name}[$j]);
$remove_ins{$k}++; # store which insertions need to be removed
$complex_event_flag++;
# PRINT INSERTION COMPONENT TO FA
if ((exists $parameters{'I'}) && ($ins_length{$name}[$k] >= $insthreshold)) {
my $ins_seq = join('',@{$seq{$name}}[$ins_starts{$name}[$k] .. $ins_ends{$name}[$k]]);
print INSERTIONS ">$name $ins_length{$name}[$k] from complex event ";
print INSERTIONS "-$del_length{$name}[$j]+$ins_length{$name}[$k] ";
if ($ucscpos eq "no") { print INSERTIONS ($ins_starts{$name}[$k]+1)." .. ".($ins_ends{$name}[$k]+1)." in "; }
else { print INSERTIONS "$ins_starts{$name}[$k] .. ".($ins_ends{$name}[$k]+1)." in "; }
print INSERTIONS "$datafile\n$ins_seq\n";
}
}
if ($ins_starts{$name}[$k] == $next_pos) { # deletion then insertion?
# COMPLEX EVENT
# store complex event characters
push (@{$complex_types{$name}}, "-$del_length{$name}[$j]+$ins_length{$name}[$k]");
push (@{$complex_starts{$name}}, $del_starts{$name}[$j]);
push (@{$complex_ends{$name}}, $ins_ends{$name}[$k]);
push (@{$Cdel_length{$name}}, $del_length{$name}[$j]);
$remove_ins{$k}++; # store which insertions need to be removed
$complex_event_flag++;
# PRINT INSERTION COMPONENT TO FA
if ((exists $parameters{'I'}) && ($ins_length{$name}[$k] >= $insthreshold)) {
my $ins_seq = join('',@{$seq{$name}}[$ins_starts{$name}[$k] .. $ins_ends{$name}[$k]]);
print INSERTIONS ">$name $ins_length{$name}[$k] from complex event ";
print INSERTIONS "-$del_length{$name}[$j]+$ins_length{$name}[$k] ";
if ($ucscpos eq "no") { print INSERTIONS ($ins_starts{$name}[$k]+1)." .. ".($ins_ends{$name}[$k]+1)." in "; }
else { print INSERTIONS "$ins_starts{$name}[$k] .. ".($ins_ends{$name}[$k]+1)." in "; }
print INSERTIONS "$datafile\n$ins_seq\n";
}
}
}
unless (defined $complex_event_flag) {
push (@new_del_starts, $del_starts{$name}[$j]);
push (@new_del_ends, $del_ends{$name}[$j]);
push (@new_del_length, $del_length{$name}[$j]);
}
}
for (my $i=0; $i < @{$ins_starts{$name}}; $i++) {
unless (exists $remove_ins{$i}) {
push (@new_ins_starts, $ins_starts{$name}[$i]);
push (@new_ins_ends, $ins_ends{$name}[$i]);
push (@new_ins_length, $ins_length{$name}[$i]);
}
}
@{$del_starts{$name}} = @new_del_starts; # remove complex events from dels
@{$del_ends{$name}} = @new_del_ends;
@{$del_length{$name}} = @new_del_length;
@{$ins_starts{$name}} = @new_ins_starts; # remove complex events from ins
@{$ins_ends{$name}} = @new_ins_ends;
@{$ins_length{$name}} = @new_ins_length;
}
if ($alphaU{$name} == 0) { next; } # skip sequences which do not align with ref (see also below)
# POOL ALL INSERTION LENGTHS FOR ALIGNMENT
if (defined $ins_length{$name}[0]) { @allins_lengths = (@allins_lengths,@{$ins_length{$name}}); }
# POOL ALL DELETION LENGTHS FOR ALIGNMENT
if (defined $del_length{$name}[0]) { @alldel_lengths = (@alldel_lengths,@{$del_length{$name}}); }
# POOL ALL COMPLEX TYPES & LENGTHS
if (defined $complex_types{$name}[0]) {
@allcomplex_types = (@allcomplex_types,@{$complex_types{$name}});
@allCdel_lengths = (@allCdel_lengths,@{$Cdel_length{$name}});
}
# PRINT LENGTHS TO LENGTH FILE
#
my @non_unique_seq = @{$seq{$name}}[(keys %shared_pos)]; # calculate length from summary
my $non_unique_length = grep (/[a-z]/i, @non_unique_seq);
print LENGTHS "$name\t$initial_length{$name}\t$final_length{$name}\t$insertion_length{$name}\t$non_unique_length\t$fewseq_length{$name}";
# calculate length from summary
$alphaI{$name} = ($final_length{$name} - $insertion_length{$name} + $initial_length{$name}) / 2;
$alphaIJustin{$name} = ($final_length{$name} + $initial_length{$name}) / 2;
print LENGTHS "\t$alphaI{$name}\t$alphaU{$name}";
# calculate length from summary
my $test;
if ($getuniqueatshared eq "no") {
$test = $final_length{$name} - $insertion_length{$name} - $non_unique_length - $fewseq_length{$name};
}
else { $test = $final_length{$name} - $insertion_length{$name} - $fewseq_length{$name}; }
if ($test ne $alphaU{$name}) { # BUG CHECK
print "WARNING. $name. length test alphaU on fly $alphaU{$name} not equal to length from summaries $test.\nCheck for bugs\n";
}
foreach my $codon_pos (1,2,3,0) {
if (defined $ps_length{"$name$codon_pos"}) {
print LENGTHS "\t".$alphaU{"$name**$codon_pos"};
}
else { print LENGTHS "\t0"; }
}
foreach my $base (@bases) {
print LENGTHS "\t".$alphaU{"$name$base"};
}
if (($getuniqueatshared eq "no") && (exists $parameters{'c'})) {
foreach my $type (@neighbour_bases) {
print LENGTHS "\t".$alphaU{"$name$type"};
}
}
if ($getuniqueatshared eq "yes") {
if (exists $parameters{'c'}) {
foreach my $type (@neighbour_changes) {
print LENGTHS "\t".$alphaU{"$name$type"};
}
}
else {
foreach my $change (@changes,"?") {
print LENGTHS "\t".$alphaU{"$name$change"};
}
}
}
print LENGTHS "\n";
}
# SUMMARISE CODON POSITIONS OF NON_UNIQUE SITES
#
my $first = 0; my $second = 0; my $third = 0; my @other;
my @nonunique_pos = sort { $a <=> $b } (keys %shared_pos);
foreach my $codon_position (@index[@nonunique_pos]) {
if ($codon_position == 1) { $first++; }
elsif ($codon_position == 2) { $second++; }
elsif ($codon_position == 3) { $third++; }
else { push (@other, $codon_position); }
}
# IDENTIFY SHARED INSERTIONS, DELETIONS AND COMPLEX EVENTS
my $ins_class_ref = identify_shared(\%ins_starts,\%ins_ends,\%ins_length,"insertion");
my $del_class_ref = identify_shared(\%del_starts,\%del_ends,\%del_length,"deletion");
%ins_class = %$ins_class_ref;
%del_class = %$del_class_ref;
unless (exists $parameters{'C'}) {
my $complex_class_ref = identify_shared(\%complex_starts,\%complex_ends,\%Cdel_length,"complex");
%complex_class = %$complex_class_ref;
}
# PRINT SUBSTITUTIONS & count totals for unique codon positions
#
my $ufirst = 0; my $usecond = 0; my $uthird = 0; my @uother;
my $uins = 0; my $udels = 0; my $ucomplex = 0;
foreach my $name (@names) {
if ($alphaU{$name} == 0) { # skip sequences which do not align with ref (see also above)
print "WARNING: Skipping $name because it has no sequence that aligns with the reference sequence.\n";
next;
}
for (my $i=0; $i < @{$ins_starts{$name}}; $i++) { # PRINT INSERTIONS
if ($ucscpos eq "no") { print OUT "$name\t$ins_class{$name}[$i]\t$ins_length{$name}[$i]\t".($ins_starts{$name}[$i]+1)." .. ".($ins_ends{$name}[$i]+1)."\tn/a\n"; }
else { print OUT "$name\t$ins_class{$name}[$i]\t$ins_length{$name}[$i]\t$ins_starts{$name}[$i] .. ".($ins_ends{$name}[$i]+1)."\tn/a\n"; }
if ($ins_class{$name}[$i] !~ /shared/) { $uins++; }
if ((exists $parameters{'I'}) && ($ins_length{$name}[$i] >= $insthreshold)) { # fasta file if requested
my $ins_seq = join('',@{$seq{$name}}[$ins_starts{$name}[$i] .. $ins_ends{$name}[$i]]);
if ($ucscpos eq "no") { print INSERTIONS ">$name $ins_length{$name}[$i] ".($ins_starts{$name}[$i]+1)." .. ".($ins_ends{$name}[$i]+1)." in $datafile\n$ins_seq\n"; }
else { print INSERTIONS ">$name $ins_length{$name}[$i] $ins_starts{$name}[$i] .. ".($ins_ends{$name}[$i]+1)." in $datafile\n$ins_seq\n"; }
}
}
for (my $i=0; $i < @{$del_starts{$name}}; $i++) { # PRINT DELETIONS
if ($ucscpos eq "no") { print OUT "$name\t$del_class{$name}[$i]\t$del_length{$name}[$i]\t".($del_starts{$name}[$i]+1)." .. ".($del_ends{$name}[$i]+1)."\tn/a\n"; }
else { print OUT "$name\t$del_class{$name}[$i]\t$del_length{$name}[$i]\t$del_starts{$name}[$i] .. ".($del_ends{$name}[$i]+1)."\tn/a\n"; }
if ($del_class{$name}[$i] !~ /shared/) { $udels++; }
}
unless (exists $parameters{'C'}) { # PRINT COMPLEX EVENTS
if (defined @{$complex_starts{$name}}) {
for (my $i=0; $i < @{$complex_starts{$name}}; $i++) {
if ($ucscpos eq "no") { print OUT "$name\t$complex_class{$name}[$i]\t$complex_types{$name}[$i]\t".($complex_starts{$name}[$i]+1)." .. ".($complex_ends{$name}[$i]+1)."\tn/a\n"; }
else { print OUT "$name\t$complex_class{$name}[$i]\t$complex_types{$name}[$i]\t$complex_starts{$name}[$i] .. ".($complex_ends{$name}[$i]+1)."\tn/a\n"; }
if ($complex_class{$name}[$i] !~ /shared/) { $ucomplex++; }
}
}
}
if (defined $unique_ps{$name}[0]) { # PRINT UNIQUE POINT SUBS
for (my $i=0; $i < @{$unique_ps{$name}}; $i++) {
if ($ucscpos eq "no") { print OUT "$name\tunique_ps\t$unique_ps_type{$name}[$i]\t".($unique_ps{$name}[$i]+1)." .. ".($unique_ps{$name}[$i]+1)."\t$index[$unique_ps{$name}[$i]]\n"; }
else { print OUT "$name\tunique_ps\t$unique_ps_type{$name}[$i]\t$unique_ps{$name}[$i] .. ".($unique_ps{$name}[$i]+1)."\t$index[$unique_ps{$name}[$i]]\n"; }
if ($index[$unique_ps{$name}[$i]] == 1) { $ufirst++; }
elsif ($index[$unique_ps{$name}[$i]] == 2) { $usecond++; }
elsif ($index[$unique_ps{$name}[$i]] == 3) { $uthird++; }
else { push ( @uother, $index[$unique_ps{$name}[$i]]); }
}
}
}
# PRINT SUMMARY OF NON-UNIQUE POINT SUBS TO FILE
#
if ((exists $parameters{'s'}) && ((keys %shared_pos) > 0)) {
open NONUNIQUE, ">$nonuniquefile" or die "couldn't open $nonuniquefile : $!";
print NONUNIQUE "# ".@nonunique_pos." non-unique positions, ".@names." sequences in $datafile alignment\n";
print NONUNIQUE "# ";
foreach (@nonunique_pos) { print NONUNIQUE ($_+1)." "; } # using normal (base-1) coordinates for output fasta file, because output does not give a range so ucsc is less applicable
print NONUNIQUE "\n";
print NONUNIQUE "# @index[@nonunique_pos]\n";
print NONUNIQUE "# codon position totals.\t1st: $first\t2nd: $second\t3rd: $third\tother: ";
if (@other > 0) { print NONUNIQUE @other." (@other)\n"; }
else { print NONUNIQUE "0\n"; }
foreach my $name (@refseqnames,@names) {
print NONUNIQUE ">$name\n";
foreach my $pos (@nonunique_pos) {
print NONUNIQUE "$seq{$name}[$pos]";
}
print NONUNIQUE "\n";
}
}
# PRINT SUMMARY OF SITES WHERE THERE WAS A UNIQUE SUB AND THE ANCESTOR WAS AMBIGUOUS
#
unless (($getuniqueatshared eq "no") || (keys %ambanc == 0)) {
open AMBANCESTOR, ">$ambancfile" or die "couldn't open $ambancfile : $!";
my @ambanc_pos = sort { $a <=> $b } (keys %ambanc);
print AMBANCESTOR "position\tNo.ofSeqsToCheck\tname\tbase\tpossible_ancestors\tSEE BELOW FOR A SUMMARY OF ALL DATA FOR JUST THESE SITES\n";
foreach my $pos (@ambanc_pos){
my @ambnames = @{$ambanc{$pos}};
print AMBANCESTOR ($pos+1)."\t".@ambnames; # using normal (base-1) coordinates for output fasta file, because output does not give a range so ucsc is less applicable
foreach my $name (@ambnames) { print AMBANCESTOR "\t$name\t$seq{$name}[$pos]\t$ambancestors{$pos}"; }
print AMBANCESTOR "\n";
}
print AMBANCESTOR "\nALL SEQS FOR SITES THAT NEED MANUAL CHECKING:\n";
foreach my $pos (@ambanc_pos) { print AMBANCESTOR "\t$pos"; }
print AMBANCESTOR "\n";
foreach my $name (@names) {
print AMBANCESTOR "$name";
foreach my $pos (@ambanc_pos) { print AMBANCESTOR "\t$seq{$name}[$pos]"; }
print AMBANCESTOR "\n";
}
}
# SUMMARY TO STDOUT
#
if (@allins_lengths > 0) {
print "\nNo. of unique insertions :\t$uins\n";
print "No. of all insertions :\t\t".@allins_lengths."\n";
print "Lengths of all insertions :\t@allins_lengths\n";
}
else { print "\nNo insertions.\n"; }
if (@alldel_lengths > 0) {
print "\nNo. of unique deletions :\t$udels\n";
print "No. of all deletions :\t\t".@alldel_lengths."\n";
my $complete = numeric(@alldel_lengths);
print "No. of all complete deletions :\t$complete\n";
print "Lengths of all deletions :\t@alldel_lengths\n";
}
else { print "\nNo deletions.\n"; }
unless (exists $parameters{'C'}) {
if (@allcomplex_types > 0) {
print "\nNo. of unique complex events :\t\t$ucomplex\n";
print "No. of all complex events :\t\t".@allcomplex_types."\n";
my $complete = numeric(@allCdel_lengths);
print "No. of all complete complex events :\t$complete\n";
print "Lengths of all complex events :\t\t@allcomplex_types\n";
}
else { print "\nNo complex events.\n"; }
}
print "\nNon-unique point sub positions :\t".(keys %shared_pos)."\n";
print "Codon positions of non-unique sites :\t$first : $second : $third";
if (@other > 0) { print "\tothers: ".@other."\n"; }
else { print "\n"; }
print "No. of unique point subs :\t\t$unique_ps_count\n";
print "Codon positions of unique sites :\t$ufirst : $usecond : $uthird";
if (@other > 0) { print "\tothers: ".@uother."\n"; }
else { print "\n"; }
if (keys %ambanc > 0) {
print "Unique sub sites with >1 poss ancestor:\t".(keys %ambanc)." (check these & adjust all alphaUs).\n";
}
print "\n";
# CALCULATE RELATIVE RATES OF DIFFERENT TYPES OF EVENT
#
# THIS HAS BEEN CHECKED BY RUNNING insertion THROUGH
# relative_rate. GET THE SAME ANSWER AFTER REMOVING
# j SUBTRACTIONS & ADDITIONS
my ($lambdaI) = insertion(\@names,\%ins_length,\%unique_ps,\%alphaI,\%alphaU);
print "Relative rate of insertion (lambdaI) :\t$lambdaI ins per point sub\n";
my ($lambdaIJustin) = insertion(\@names,\%ins_length,\%unique_ps,\%alphaIJustin,\%alphaU);
print "Using Blumenstiel et al 02 :\t\t$lambdaIJustin ins per point sub\n\n";
my $maxdel_length = max(@alldel_lengths); # GRAB MAX DEL LENGTH
# ESTIMATE RELATIVE RATE OF DELS
my ($lambdaD,$lambdaDJustin) = relative_rate(\@names,$maxdel_length,\%del_length,\%unique_ps,\%final_length,\%initial_length,\%insertion_length,\%alphaU);
# PRINT RELATIVE RATE RATE OF DELS
if ($lambdaD =~ /</) { print "No deletions. Probably < relative-rate given 1 event (1bp long).\n"; }
print "Relative rate of deletion (lambdaD) :\t$lambdaD dels per point sub\n";
print "Using Blumenstiel et al 02 :\t\t$lambdaDJustin dels per point sub\n\n";
unless (exists $parameters{'C'}) {
my $maxCdel_length = max(@allCdel_lengths); # GRAB COMPLEX EVENT MAX DEL LENGTH
# ESTIMATE RELATIVE RATE OF COMPLEX EVENTS
my ($lambdaC,$lambdaCJustin) = relative_rate(\@names,$maxCdel_length,\%Cdel_length,\%unique_ps,\%final_length,\%initial_length,\%insertion_length,\%alphaU);
if ($lambdaC =~ /</) { print "No complex events. Probably < relative-rate given 1 event (1bp long).\n"; }
print "Relative rate of complex events (lambdaC) :\t$lambdaC complex events per point sub\n";
print "Using Blumenstiel et al 02 :\t\t\t$lambdaCJustin complex events per point sub\n";
}
#################
## SUBROUTINES ##
#################
# IN ORDER OF APPEARANCE #
## READ IN ALIGNMENT FROM NEXUS FORMAT
#
sub nexus {
my $datafile = shift;
my (%seq, @names, %seen);
my $length = 0;
open DATA, "<$datafile" or die "couldn't open $datafile : $!";
while (<DATA>) {
if (/^(\S+)\s+([$seq_recog]+)\s*?\[*?\d*?\]*?$/mi) {
my $name = $1;