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trevolver.pl
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trevolver.pl
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#! /usr/bin/perl
#########################################################################################
# Perl script to simulate sequence evolution on a BIFURCATING TREE provided in Newick
# format with a user-provided TRINUCLEOTIDE (64 x 4) rate matrix, such as that described
# in SLiM. This allows non-reversible context-dependent evolution with back mutation.
# OUTPUTS: mutation data, VCF file
#########################################################################################
# EXAMPLES
#########################################################################################
# [FORMAT] trevolver.pl --tree=<newick>.txt --seed_sequence=<seed>.fasta --rate_matrix=<64x4>.txt --branch_unit=<#> --random_seed=<#>
#########################################################################################
# [EXAMPLE 1] trevolver.pl --tree=../EXAMPLE_INPUT/tree_6taxa.txt --seed_sequence=../EXAMPLE_INPUT/seed_sequence.fa --rate_matrix=../EXAMPLE_INPUT/mutation_CpGx20.txt --vcf_output=example1.vcf --branch_unit=10000 > example1.txt
#########################################################################################
# [EXAMPLE 2] trevolver.pl --tree=../EXAMPLE_INPUT/tree_6taxa.txt --seed_sequence=../EXAMPLE_INPUT/seed_sequence.fa --rate_matrix=../EXAMPLE_INPUT/mutation_CpGx20.txt --vcf_output=example2.vcf --branch_unit=10000 --burn_in=1000 > example2.txt
#########################################################################################
# [EXAMPLE 3] trevolver.pl --tree=../EXAMPLE_INPUT/tree_6taxa.txt --seed_sequence=../EXAMPLE_INPUT/seed_sequence.fa --rate_matrix=../EXAMPLE_INPUT/mutation_CpGx20.txt --branch_unit=144740 --track_mutations --tracked_motif=CG --vcf_output=example3.vcf > example3.txt
#########################################################################################
# [EXAMPLE 4] trevolver.pl --tree=../EXAMPLE_INPUT/tree_10taxa.txt --seed_sequence=../EXAMPLE_INPUT/seed_sequence.fa --rate_matrix=../EXAMPLE_INPUT/mutation_CpGx20.txt --branch_unit=144740 --track_mutations --tracked_motif=CG --vcf_output=example4.vcf --outgroups=2 > example4.txt
#########################################################################################
# [EXAMPLE 5] trevolver.pl --tree=../EXAMPLE_INPUT/tree_1taxon.txt --seed_sequence=../EXAMPLE_INPUT/seed_sequence.fa --rate_matrix=../EXAMPLE_INPUT/mutation_equal.txt --vcf_output=example5.vcf --branch_unit=1 > example5.txt
#########################################################################################
# [EXAMPLE 6] trevolver.pl --tree=../EXAMPLE_INPUT/tree_7taxa.txt --seed_sequence=../EXAMPLE_INPUT/seed_sequence.fa --rate_matrix=../EXAMPLE_INPUT/mutation_equal.txt --branch_unit=144740 --random_seed=123456789 --tracked_motif=CG --track_mutations --vcf_output=example6.vcf --outgroups=2 --burn_in=500 --suppress_seed_seq --suppress_consensus_seq --verbose > example6.txt
#########################################################################################
# [EXAMPLE 7] trevolver.pl --tree=../EXAMPLE_INPUT/tree_7taxa.txt --seed_sequence=../EXAMPLE_INPUT/seed_sequence.fa --rate_matrix=../EXAMPLE_INPUT/mutation_CpGx20.txt --branch_unit=1447
#########################################################################################
#########################################################################################
# Copyright (C) 2019 Chase W. Nelson
# DATE CREATED: June 2019
# AUTHOR: Chase W. Nelson
# CONTACT1: cnelson@amnh.org
# AFFILIATION: Sackler Institute for Comparative Genomics, American Museum of Natural
# History, New York, NY 10024, USA
# ACKNOWLEDGMENTS: written by C.W.N. with support from a Gerstner Scholars Fellowship from
# the Gerstner Family Foundation at the American Museum of Natural History, New York.
#########################################################################################
use strict;
use List::Util qw(max sum);
use Getopt::Long;
STDOUT->autoflush(1);
# Get the time
my $time1 = time;
my $local_time1 = localtime;
my @commands = @ARGV;
#########################################################################################
# INITIALIZE INPUT VARIABLES
my $tree; # file containing bifurcating evolutionary tree with branch lengths
my $seed_sequence; # file containing starting (seed) sequence at tree root, to be evolved
my $rate_matrix; # file containing 64 x 4 tab-delimited rate matrix in alphabetical order. First row values for: AAA>AAA\tAAA>ACA\tAAA>AGA\tAAA>ATA\n
my $branch_unit; # branch lengths will be multiplied by this value and rounded up to the nearest integer to determine number of generations
my $random_seed; # integer with which to seed the random number generator
my $tracked_motif;
my $track_mutations;
my $vcf_output;
my $excluded_taxa;
my $outgroups;
my $burn_in;
my $suppress_input_seq;
my $suppress_seed_seq;
my $suppress_MRCA_seq;
my $suppress_consensus_seq;
my $suppress_MUTATION;
my $verbose;
# Get user input, if given. If a Boolean argument is passed, its value is 1; else undef
GetOptions( "tree=s" => \$tree,
"seed_sequence=s" => \$seed_sequence,
"rate_matrix=s" => \$rate_matrix,
"branch_unit=f" => \$branch_unit,
"random_seed=i" => \$random_seed,
"tracked_motif=s" => \$tracked_motif,
"track_mutations" => \$track_mutations,
"vcf_output=s" => \$vcf_output,
"excluded_taxa=s" => \$excluded_taxa,
"outgroups=i" => \$outgroups,
"burn_in=i" => \$burn_in,
"suppress_input_seq" => \$suppress_input_seq,
"suppress_seed_seq" => \$suppress_seed_seq,
"suppress_MRCA_seq" => \$suppress_MRCA_seq,
"suppress_consensus_seq" => \$suppress_consensus_seq,
"suppress_MUTATION" => \$suppress_MUTATION,
"verbose" => \$verbose
)
or print_usage_message("### WARNING: Error in command line arguments (option misspelled?). trevolver terminated.");
#print "\nvcf_output=$vcf_output\n"; # the value of a called flag is 1
unless(-f "$tree") {
my $specific_warning = "### WARNING: A valid --tree option must be provided.";
print_usage_message($specific_warning);
}
unless(-f "$seed_sequence") {
my $specific_warning = "### WARNING: A valid --seed_sequence option must be provided.";
print_usage_message($specific_warning);
}
unless(-f "$rate_matrix") {
my $specific_warning = "### WARNING: A valid --rate_matrix option must be provided.";
print_usage_message($specific_warning);
}
unless($branch_unit =~ /\d/) {
my $specific_warning = "### WARNING: A valid --branch_unit option must be provided.";
print_usage_message($specific_warning);
}
if (-f "$vcf_output" || $vcf_output =~ /^\-/) {
my $specific_warning = "### WARNING: A valid --vcf_output file name must be provided (or already exists).";
print_usage_message($specific_warning);
}
#if ($excluded_taxa && $outgroups) {
# my $specific_warning = "### WARNING: The --excluded_taxa and --outgroups options are mutually exclusive; they cannot both be called.";
# print_usage_message($specific_warning);
#}
my $working_directory = `pwd`;
chomp($working_directory);
#print "\nworking_directory=$working_directory\n"; # does NOT include a trailing forward slash, but does end in a newline
##########################################################################################
# Extract tree file prefix
my $file_prefix;
if($tree =~/\/*([^\/]+)\..+/) {
$file_prefix = $1;
} else {
$file_prefix = 'trevolver_input';
}
unless ($vcf_output =~ /\w/) {
$vcf_output = $file_prefix . ".vcf";
}
##########################################################################################
# Store the tree(s)
open(IN_TREE, "$tree") or die "Could not open file $tree\n";
my $tree = '';
# Store first tree in file
while(<IN_TREE>) {
chomp($_);
my $line = $_;
if($line =~ /^\(/) { # line starts with an opening parentheses
if($line =~ /\);$/) { # line ends with a closing parentheses and semicolon
$tree = $line;
last;
}
}
}
close IN_TREE;
if($tree eq '') {
my $specific_warning = "### WARNING: NO TREE IN TREE FILE. Must begin with '(' and end with ');'.";
print_usage_message($specific_warning);
}
# Trim end of tree if ';' or whitespace
#print "\nTREE:\n$tree\n\n";
while(substr($tree, -1) =~ /[\s\;]/) {
chop($tree);
}
#print "\nPROCESSED TREE:\n$tree\n\n";
if($tree =~ /(\,[a-zA-Z0-9\.\-\|\:\']+\,)/) {
my $specific_warning = "### WARNING: TREE MUST BE STRICTLY BIFURCATING BUT CONTAINS A POLYTOMY: $1";
print_usage_message($specific_warning);
}
if($tree =~ /(\(\,[^\(]*\,\()/) {
my $specific_warning = "### WARNING: TREE MUST BE STRICTLY BIFURCATING BUT CONTAINS A POLYTOMY: $1";
print_usage_message($specific_warning);
}
if($tree =~ /(\)\d+)/) {
my $specific_warning = "### WARNING: CURRENTLY, SUPPORT VALUES ARE NOT ALLOWED BUT ARE PRESENT: $1";
print_usage_message($specific_warning);
}
if($tree =~ /(\)\[)/) {
my $specific_warning = "### WARNING: CURRENTLY, SUPPORT VALUES ARE NOT ALLOWED BUT ARE PRESENT: $1";
print_usage_message($specific_warning);
}
if($tree =~ /(\],)/) {
my $specific_warning = "### WARNING: CURRENTLY, SUPPORT VALUES ARE NOT ALLOWED BUT ARE PRESENT: $1";
print_usage_message($specific_warning);
}
my $tree_opening_paren_count = ($tree =~ s/\(/\(/g);
my $tree_closing_paren_count = ($tree =~ s/\)/\)/g);
unless($tree_opening_paren_count > 0 && $tree_opening_paren_count == $tree_closing_paren_count) {
my $specific_warning = "### WARNING: TREE MUST CONTAIN EQUAL NUMBERS OF OPENING (now $tree_opening_paren_count\) AND CLOSING " .
"(now $tree_closing_paren_count\) PARENTHESES.";
print_usage_message($specific_warning);
}
##########################################################################################
# Read in the seed sequence from the fasta file
my $seed_seq = '';
my $seq_num = 0;
open(IN_FASTA, "$seed_sequence") or die "Could not open file $seed_sequence\n";
if($verbose) {
print "\n################################################################################";
print "\nRecording seed sequence data from $seed_sequence...\n";
}
my $fasta_header = '';
while(<IN_FASTA>) {
chomp;
if(/>([\S]+)\s*/) {
$fasta_header = $1;
if($seq_num == 0) {
$seq_num ++;
} else {
last;
}
} else {
$seed_seq .= $_;
}
}
close IN_FASTA;
$seed_seq = uc($seed_seq);
$seed_seq =~ tr/U/T/;
unless($seed_seq =~ /[ACGT]/ && length($seed_seq) >= 3) {
my $specific_warning = "### WARNING: Sequence does not contain more than two nucleotides (A, C, G, T).";
print_usage_message($specific_warning);
}
my $seed_seq_length = length($seed_seq);
##########################################################################################
# Read in the rate matrix
my %rate_matrix;
my @ordered_trinucleotides = qw/AAA AAC AAG AAT
ACA ACC ACG ACT
AGA AGC AGG AGT
ATA ATC ATG ATT
CAA CAC CAG CAT
CCA CCC CCG CCT
CGA CGC CGG CGT
CTA CTC CTG CTT
GAA GAC GAG GAT
GCA GCC GCG GCT
GGA GGC GGG GGT
GTA GTC GTG GTT
TAA TAC TAG TAT
TCA TCC TCG TCT
TGA TGC TGG TGT
TTA TTC TTG TTT/;
# ^ each of those corresponds to ONE ROW of the rate matrix
open(IN_RATE_MATRIX, "$rate_matrix") or die "Could not open file $rate_matrix\n";
if ($verbose) {
print "\n################################################################################";
print "\nRecording rate matrix from $rate_matrix...\n";
}
my $row_index = 0;
my $max_whitespaces = 0;
while(<IN_RATE_MATRIX>) {
chomp;
my $line = $_;
my $num_whitespaces = ($line =~ s/(\s+)/$1/g);
if($num_whitespaces > $max_whitespaces) { $max_whitespaces = $num_whitespaces }
if (/([0-9\.eE\-]+)\s+([0-9\.eE\-]+)\s+([0-9\.eE\-]+)\s+([0-9\.eE\-]+)$/) { # in case there's a header or a column of names in front
$rate_matrix{$ordered_trinucleotides[$row_index]}->{'A'} = $1;
$rate_matrix{$ordered_trinucleotides[$row_index]}->{'C'} = $2;
$rate_matrix{$ordered_trinucleotides[$row_index]}->{'G'} = $3;
$rate_matrix{$ordered_trinucleotides[$row_index]}->{'T'} = $4;
$row_index++;
}
}
close IN_RATE_MATRIX;
unless($row_index == 64 || $max_whitespaces <= 5) { # i.e., one more than the last real index
my $specific_warning = "### WARNING: There must be 64 rows X 4 columns of data in the rate matrix.";
print_usage_message($specific_warning);
}
if ($verbose) { print "\n" }
print "################################################################################".
"\n## ##".
"\n## Evolution On Tree Using Custom Rates Initiated! ##".
"\n## ##".
"\n################################################################################\n";
print "\nAnalysis initiated at local time $local_time1\n";
print "\nCOMMAND: trevolver.pl @commands\n";
##########################################################################################
# Generate or assign random seed value
if($random_seed) {
print "\nRANDOM_SEED: $random_seed\n";
srand($random_seed);
} else {
$random_seed = srand(time ^ $$ ^ unpack "%32L*", `ps wwaxl | gzip`); # (Programming Perl, p. 955)
print "\nRANDOM_SEED: $random_seed\n";
}
##########################################################################################
# Read in the excluded taxa names
my %excluded_taxa_names;
if ($excluded_taxa) {
if (-f "$excluded_taxa") {
open(IN_EXCLUDED_TAXA, "$excluded_taxa");
if($verbose) {
print "\n################################################################################";
print "\nRecording excluded taxa from $excluded_taxa...\n";
}
while(<IN_EXCLUDED_TAXA>) {
chomp;
my $line = $_;
if($line =~ /\w/) {
my @this_line_taxa = split(/\,\s*/, $line);
print "\nEXCLUDED TAXA NAMES: ";
my $excluded_taxa_line = '';
foreach (@this_line_taxa) {
$excluded_taxa_names{$_} = 1;
$excluded_taxa_line .= "$_\,";
}
chop($excluded_taxa_line);
print "$excluded_taxa_line\n";
}
}
close IN_EXCLUDED_TAXA;
} else {
print "\n### WARNING: could not open file $excluded_taxa\. Excluding no taxa.\n";
}
}
##########################################################################################
# STORE THE TREE AS A MULTIDMINENSIONAL HASH
if ($verbose) {
print "\n################################################################################\n";
print "Recursively trevolving sequences from the root...\n";
}
#my %tree;
print "\nTREE: $tree\n";
##########################################################################################
# IDENTIFY AND CHARACTERIZE OUTGROUPS
##########################################################################################
my @outgroup_data; # [0] generation MRCA [1-n] outgroup names
# Obtain OUTGROUP NAMES and the GENERATION in which the MRCA of the ingroup lived.
if ($outgroups) {
if ($verbose) { print "\n### OUTGROUP IDENTIFICATION COMMENCING...\n" }
# PASS: tree, generations elapsed, curr_outgroup_count, outgroup_names
@outgroup_data = determine_outgroup_data($tree, 0, 0, '');
# RETURNS: subtree with MRCA root; generation of MRCA; outgroups 1-n
}
my $MRCA_subtree;
my $MRCA_generation;
if (@outgroup_data) {
$MRCA_subtree = shift(@outgroup_data);
$MRCA_generation = shift(@outgroup_data);
print "\nMRCA_GENERATION: $MRCA_generation\n";
print "\nMRCA_SUBTREE: $MRCA_subtree\n";
print "\nOUTGROUPS: @outgroup_data\n";
} else {
undef($outgroups);
}
#@outgroup_data now contains only outgroup names
if ($outgroups) {
foreach (@outgroup_data) {
$excluded_taxa_names{$_} = 1;
}
}
##########################################################################################
##########################################################################################
##########################################################################################
# BURN-IN
##########################################################################################
my $num_burn_in_mutations = 0;
my $input_seq = '';
if ($burn_in) {
$input_seq = $seed_seq;
$seed_seq = burn_in($seed_seq, $burn_in);
if ($verbose) { print "BURN-IN INVOLVED $num_burn_in_mutations MUTATIONS\n" }
}
if ($input_seq ne '') {
unless($suppress_input_seq) { print "\nINPT_SEQUENCE: $input_seq\n" }
}
unless($suppress_seed_seq) { print "\nSEED_SEQUENCE: $seed_seq\n" }
##########################################################################################
##########################################################################################
##########################################################################################
# THE SIMULATION: recursive evolution approach using the subroutine evolve_branch()
##########################################################################################
my $num_mutations = 0;
my $total_branch_length = 0;
my $root_to_tip_length = 0;
my %taxa_histories;
my %generational_histories;
my $node_id = 0;
my %mutated_sites;
my %MRCA_mutation_history;
my $MRCA_seq;
my $MRCA_node_id;
if ($verbose) { print "\n###EVOLUTION ON TREE COMMENCING...\n" }
evolve_branch($tree, 0, 0, 'n0=root,');
##########################################################################################
##########################################################################################
# Output consensus and MRCA information
if ($outgroups) {
print "\nMRCA_NODE_ID: $MRCA_node_id\n";
unless($suppress_MRCA_seq) { print "\nMRCA_SEQUENCE: $MRCA_seq\n" }
}
##########################################################################################
##########################################################################################
### VCF output
##########################################################################################
##########################################################################################
if ($vcf_output =~ /\w+/) {
chomp($vcf_output);
my (undef, undef, undef, $day, $month, $year) = localtime;
$year = $year + 1900;
$month += 1;
if (length($month) == 1) { $month = "0$month" }
if (length($day) == 1) { $day = "0$day" }
my $today = "$year$month$day";
if ($vcf_output =~ /\//) { # a path was provided
my $vcf_output_dir = $vcf_output;
$vcf_output_dir =~ s/\/[^\/]+$//;
if (-d "$vcf_output_dir") {
if (-f "$vcf_output") { # file already exist?
my $new_vcf_output = "$working_directory\/$file_prefix\_$random_seed\.vcf";
print "\nVCF_OUTPUT_FILE: SPECIFIED FILE ALREADY EXISTS, NEW FILE NAME: $new_vcf_output.\n";
$vcf_output = $new_vcf_output;
} else { # else we're good to go
print "\nVCF_OUTPUT_FILE: $vcf_output.\n";
}
} else {
my $new_vcf_output = "$working_directory\/$file_prefix\_$random_seed\.vcf";
print "\nVCF_OUTPUT_FILE: NON-EXISTENT DIRECTORY SPECIFIED, NEW FILE NAME: $new_vcf_output.\n";
$vcf_output = $new_vcf_output;
}
} else {
print "\nVCF_OUTPUT_FILE: $working_directory\/$vcf_output.\n";
$vcf_output = "$working_directory\/$vcf_output";
}
# Determine site frequency data
my %site_to_alleles; # made only for the INGROUP
my %site_to_outgroups; # made only for the OUTGROUP(S)
my $num_taxa = 0;
my $num_outgroups = 0;
# KEYS of %mutated_sites
foreach my $mutated_site (sort {$a <=> $b} keys %mutated_sites) {
my $AA = substr($seed_seq, $mutated_site - 1, 1);
# Store history
my %prev_state_1;
my %prev_state_2;
my %prev_change;
foreach my $taxon (sort {$a <=> $b} keys %taxa_histories) {
# OUTGROUP(S), if any
if ($excluded_taxa_names{$taxon}) {
if ($taxa_histories{$taxon}->{$mutated_site}) { # this taxon HAS the mutated site
my $mutation_history = $taxa_histories{$taxon}->{$mutated_site};
#print "mutation_history=$mutation_history\n";
my @mutation_history_events = split(/,/, $mutation_history);
my $extant_nt = substr($mutation_history, -1);
#print "extant_nt=$extant_nt\n";
# Store nucleotide
$site_to_outgroups{$mutated_site}->{$extant_nt}++;
foreach my $event (@mutation_history_events) { # these are ordered, but won't matter with new approach
if ($event =~ /(\d+)\-\w\>\w/) {
#91404-A>G
#868627-G>A
my $generation = $1;
$site_to_outgroups{$mutated_site}->{history}->{$generation}->{$event}++;
}
}
} else { # this taxon does NOT have the mutated site, add the ancestral (seed)
$site_to_outgroups{$mutated_site}->{$AA}++;
}
# INGROUP (or, if no outgroups, everything)
} else {
if ($taxa_histories{$taxon}->{$mutated_site}) { # this taxon HAS the mutated site
my $mutation_history = $taxa_histories{$taxon}->{$mutated_site};
#print "mutation_history=$mutation_history\n";
my @mutation_history_events = split(/,/, $mutation_history);
my $extant_nt = substr($mutation_history, -1);
#print "extant_nt=$extant_nt\n";
# Store nucleotide
$site_to_alleles{$mutated_site}->{$extant_nt}++;
foreach my $event (@mutation_history_events) { # these are ordered, but won't matter with new approach
if ($event =~ /(\d+)\-\w\>\w/) {
#91404-A>G
#868627-G>A
my $generation = $1;
$site_to_alleles{$mutated_site}->{history}->{$generation}->{$event}++;
}
}
} else { # this taxon does NOT have the mutated site, add the ancestral (seed)
$site_to_alleles{$mutated_site}->{$AA}++;
}
}
}
}
# DETERMINE CONSENSUS SEQUENCE TO PRINT
my @nts = qw/A C G T/;
my $consensus_seq = $seed_seq;
foreach my $mutated_site (sort {$a <=> $b} keys %site_to_alleles) {
my $REF = '';
my $REF_count = 0;
# Remember, if it's a tie, the first alphabetically gets it.
foreach my $nt (@nts) {
if ($site_to_alleles{$mutated_site}->{$nt} > $REF_count) {
$REF = $nt;
$REF_count = $site_to_alleles{$mutated_site}->{$nt};
}
}
# Impute into consensus sequence
substr($consensus_seq, $mutated_site - 1, 1, $REF);
}
unless($suppress_consensus_seq) { print "\nCONS_SEQUENCE: $consensus_seq\n" }
######################################################################################
# INITIATE VCF FILE
my $header_VCF = '';
open(OUT_TREVOLVER_VCF, ">>$vcf_output");
$header_VCF .= "##fileformat=VCFv4.1\n" .
"##FILTER=<ID=PASS,Description=\"All filters passed\">\n" .
"##fileDate=$today\n" .
"##reference=https://github.com/chasewnelson/trevolver\n" .
"##source=<TREVOLVER,Description=\"trevolver.pl @commands\">\n" .
"##tree=$tree\;\n" .
"##seed_sequence_file=$seed_sequence\n";
if ($input_seq ne '') {
unless($suppress_input_seq) {
$header_VCF .= "##inpt_sequence=$input_seq\n";
}
}
unless($suppress_seed_seq) {
$header_VCF .= "##seed_sequence=$seed_seq\n";
}
unless($suppress_consensus_seq) {
$header_VCF .= "##cons_sequence=$consensus_seq\n";
}
if ($outgroups) {
unless($suppress_MRCA_seq) {
$header_VCF .= "##MRCA_sequence=$MRCA_seq\n";
}
$header_VCF .= "##MRCA_generation=$MRCA_generation\n" .
"##MRCA_node_id=$MRCA_node_id\n" .
"##MRCA_subtree=$MRCA_subtree\n";
}
$header_VCF .= "##rate_matrix=$rate_matrix\n" .
"##branch_unit=$branch_unit\n" .
"##random_seed=$random_seed\n";
if ($tracked_motif) {
$header_VCF .= "##tracked_motif=$tracked_motif\n";
}
if ($burn_in) {
$header_VCF .= "##burn_in_length=$burn_in\n";
$header_VCF .= "##burn_in_mutations=$num_burn_in_mutations\n";
}
$header_VCF .= "##total_mutations=$num_mutations\n" .
"##tree_length=$total_branch_length\n" .
"##experiment_length=$root_to_tip_length\n" .
"##simulation_time=" . (time - $time1) . "\n" .
"##contig=<ID=$file_prefix,assembly=1,length=$seed_seq_length>\n" .
"##FORMAT=<ID=GT,Number=1,Type=String,Description=\"Genotype\">\n" .
"##INFO=<ID=AC,Number=A,Type=Integer,Description=\"Total number of alternate alleles in called genotypes\">\n" .
"##INFO=<ID=AF,Number=A,Type=Float,Description=\"Estimated allele frequency in the range (0,1)\">\n" .
"##INFO=<ID=NS,Number=1,Type=Integer,Description=\"Number of samples with data, equivalent to number of extant sequences\">\n" .
"##INFO=<ID=AN,Number=1,Type=Integer,Description=\"Total number of alleles in called genotypes, equivalent to number of extant sequences\">\n" .
"##INFO=<ID=DP,Number=1,Type=Integer,Description=\"Total read depth; here, equivalent to number of extant sequences\">\n" .
"##INFO=<ID=AA,Number=1,Type=String,Description=\"Ancestral Allele. AA: Ancestral allele, REF:Reference Allele, ALT:Alternate Allele\">\n" .
"##INFO=<ID=MRCA,Number=1,Type=String,Description=\"Most Recent Common Ancestor Allele of the ingroup\">\n" .
"##INFO=<ID=VT,Number=.,Type=String,Description=\"indicates what type of variant the line represents\">\n" .
"##INFO=<ID=MUTATIONS,Number=.,Type=String,Description=\"unique mutations that occurred at this site\">\n" .
"##INFO=<ID=MUTATIONS_OG,Number=.,Type=String,Description=\"unique mutations that occurred at this site in the outgroup(s)\">\n" .
"##INFO=<ID=GENERATIONS,Number=.,Type=String,Description=\"the generations at which unique mutations occurred at this site\">\n" .
"##INFO=<ID=GENERATIONS_OG,Number=.,Type=String,Description=\"the generations at which unique mutations occurred at this site in the outgroup(s)\">\n" .
"##INFO=<ID=TAXA,Number=.,Type=String,Description=\"the number of extant taxa (leaves) sharing the unique mutations that occurred at this site\">\n" .
"##INFO=<ID=TAXA_OG,Number=.,Type=String,Description=\"the number of extant taxa (leaves) sharing the unique mutations that occurred at this site among the outgroup(s)\">\n" .
"##INFO=<ID=MULTIHIT,Number=0,Type=Flag,Description=\"indicates whether a site has experienced multiple hits, i.e., more than one mutation\">\n" .
"##INFO=<ID=MULTIH_OG,Number=0,Type=Flag,Description=\"indicates whether a site has experienced multiple hits in the outgroup(s)\">\n" .
"##INFO=<ID=MULTIALLELIC,Number=0,Type=Flag,Description=\"indicates whether a site is multi-allelic\">\n" .
"##INFO=<ID=MULTIA_OG,Number=0,Type=Flag,Description=\"indicates whether a site is multi-allelic among the outgroup(s)\">\n" .
"##INFO=<ID=BACK_MUTATION,Number=0,Type=Flag,Description=\"indicates whether a site has experienced back mutation, i.e., return to a previous state via MULTIHIT\">\n" .
"##INFO=<ID=BACK_M_OG,Number=0,Type=Flag,Description=\"indicates whether a site has experienced back mutation in the outgroup(s)\">\n" .
"##INFO=<ID=RECURRENT_MUTATION,Number=0,Type=Flag,Description=\"indicates whether a site has experienced recurrent mutation, i.e., the same change occurring multiple times independently\">\n" .
"##INFO=<ID=RECURRENT_M_OG,Number=0,Type=Flag,Description=\"indicates whether a site has experienced recurrent mutation within the outgroup(s)\">\n" .
"##INFO=<ID=INVARIANT_ANCESTRAL,Number=0,Type=Flag,Description=\"indicates a site has no polymorphism in the ingroup, and that the fixed state matches the ancestral allele\">\n" .
"##INFO=<ID=INVARIANT_DERIVED,Number=0,Type=Flag,Description=\"indicates a site has no polymorphism in the ingroup, and that the fixed state matches a derived allele that resulted from mutation\">\n" .
"##INFO=<ID=NO_ANCESTRAL,Number=0,Type=Flag,Description=\"indicates that no ancestral (seed) alleles remain in the extant individuals of the ingroup\">\n" .
"##INFO=<ID=ALLELES_OG,Number=.,Type=String,Description=\"list of all alleles present in the outgroup(s)\">\n" .
"##INFO=<ID=ALLELE_COUNTS_OG,Number=.,Type=String,Description=\"list of all allele counts for alleles present in the outgroup(s), in the same order as ALLELES_OG\">\n" .
"##INFO=<ID=REF_OG,Number=.,Type=String,Description=\"the consensus (major) allele of outgroup(s), which may or may not match the AA\">\n" .
"##INFO=<ID=REF_OG_COUNT,Number=.,Type=Integer,Description=\"count of outgroup allele matching the outgroup consensus\">\n" .
"##INFO=<ID=REF_OG_AF,Number=.,Type=Float,Description=\"frequency of outgroup allele matching the outgroup consensus\">\n" .
"##INFO=<ID=OG_FIXED,Number=0,Type=Flag,Description=\"indicates that a site is fixed for one allele in the outgroup(s)\">\n" .
"##INFO=<ID=OG_DIVERGED,Number=0,Type=Flag,Description=\"indicates that one or more outgroup alleles differs from one or more ingroup alleles\">\n" .
"##INFO=<ID=OG_SHARE,Number=0,Type=Flag,Description=\"indicates one or more outgroup alleles matches one or more ingroup alleles\">\n" .
"#CHROM\tPOS\tID\tREF\tALT\tQUAL\tFILTER\tINFO\n";
print OUT_TREVOLVER_VCF "$header_VCF";
foreach my $taxon (sort {$a <=> $b} keys %taxa_histories) {
# OUTGROUP(S), if any
if ($excluded_taxa_names{$taxon}) { # exclude outgroups
$num_outgroups++; # COMEBACK -- we don't appear to be using these anymore. Consider pitching.
# INGROUP (all if no outgroups)
} else {
$num_taxa++; # COMEBACK -- will this capture ALL taxa in ALL situations? What if no history? I think yes.
}
} # end all taxa
my $out_line = '';
#my %multi_hit;
foreach my $mutated_site (sort {$a <=> $b} keys %site_to_alleles) {
#print "mutated_site=$mutated_site\n";
# AA
my $AA = substr($seed_seq, $mutated_site - 1, 1);
# MRCA ALLELE
my $MRCA_ALLELE = substr($MRCA_seq, $mutated_site - 1, 1);
#print "AA=$AA\n";
# Define REF as the major (consensus) allele
my $REF = '';
my $REF_count = 0;
my $arbitrary_REF = 0;
foreach my $nt (@nts) {
if ($site_to_alleles{$mutated_site}->{$nt} > $REF_count) {
$REF = $nt;
$REF_count = $site_to_alleles{$mutated_site}->{$nt};
$arbitrary_REF = 0;
} elsif ($site_to_alleles{$mutated_site}->{$nt} == $REF_count) {
$arbitrary_REF = 1;
}
}
#print "REF=$REF\n";
my $ALT = '';
my $AC = '';
my $AN_NS_DP = 0;
my $num_alleles = 0;
my @all_nts_present;
foreach my $nt (@nts) {
#print "nt=$nt\n";
if ($site_to_alleles{$mutated_site}->{$nt} > 0) { # here, possible that back mutation eliminates variation
#print "nt=$nt\n";
push(@all_nts_present, $nt);
$AN_NS_DP += $site_to_alleles{$mutated_site}->{$nt};
$num_alleles++;
if ($nt ne $REF) {
$ALT .= "$nt\,";
$AC .= $site_to_alleles{$mutated_site}->{$nt} . ',';
}
}
}
# This will only happen if the sample is 100% REF; print it
if ($ALT eq '' && $AC eq '') {
$ALT = "$REF\,";
$AC = "$AN_NS_DP\,";
}
chop($ALT);
chop($AC);
#print "ALT=$ALT\nAC=$AC\n";
# AFs
my $AF = '';
foreach my $nt (@nts) {
if ($nt ne $REF && $site_to_alleles{$mutated_site}->{$nt} > 0) {
my $this_AF = $site_to_alleles{$mutated_site}->{$nt} / $AN_NS_DP;
$AF .= "$this_AF\,";
}
}
# This will only happen if the sample is 100% REF; print it
if ($AF eq '') {
$AF = "1\,";
}
chop($AF);
$out_line .= "$fasta_header\t$mutated_site\t.\t$REF\t$ALT\t100\tPASS\t";
$out_line .= "AC=$AC\;AF=$AF\;AN=$AN_NS_DP\;NS=$AN_NS_DP\;DP=$AN_NS_DP\;AA=$AA\;";
if ($outgroups) { $out_line .= "MRCA=$MRCA_ALLELE\;" }
$out_line .= "VT=SNP\;";
##################################################################################
# INGROUP (SNP) INFORMATION
my $mutations = '';
my $generations = '';
my $taxa = '';
my $hits = 0;
my %prev_change;
my %prev_state_1;
my %prev_state_2;
my $back_mutation = 0;
my $recurrent_mutation = 0;
foreach my $generation (sort {$a <=> $b} keys %{$site_to_alleles{$mutated_site}->{history}}) {
#$site_to_alleles{$mutated_site}->{history}->{$generation}->{$event}++;
$hits++;
# SHOULD ONLY BE ONE EVENT PER GENERATION
foreach my $event (%{$site_to_alleles{$mutated_site}->{history}->{$generation}}) {
my $event_num = $site_to_alleles{$mutated_site}->{history}->{$generation}->{$event};
if($event =~ /(\d+)\-([\>\w+]+)/) {
my $this_mutation = $2;
$mutations .= "$2\,";
$generations .= "$1\,";
$taxa .= $event_num . ',';
my @two_states = split(/>/, $this_mutation);
my $state1 = $two_states[0];
my $state2 = $two_states[1];
# Could be recurrent/parallel
if ($prev_change{$this_mutation}) {
$recurrent_mutation++;
}
$prev_change{$this_mutation}++;
# Could be back mutation
if ($prev_state_1{$state2}) {
$back_mutation++;
}
$prev_state_1{$state1}++;
$prev_state_2{$state2}++;
}
}
}
chop($mutations);
chop($generations);
chop($taxa);
if ($mutations ne '' || $generations ne '' || $taxa ne '') {
$out_line .= "MUTATIONS=$mutations\;GENERATIONS=$generations\;TAXA=$taxa\;";
}
##################################################################################
# OUTGROUP (SUB) INFORMATION
my $mutations_out = '';
my $generations_out = '';
my $taxa_out = '';
my $hits_out = 0;
my %prev_change_out;
my %prev_state_1_out;
my %prev_state_2_out;
my $back_mutation_out = 0;
my $recurrent_mutation_out = 0;
if ($outgroups) {
foreach my $generation (sort {$a <=> $b} keys %{$site_to_outgroups{$mutated_site}->{history}}) {
$hits_out++;
# SHOULD ONLY BE ONE EVENT PER GENERATION
foreach my $event (%{$site_to_outgroups{$mutated_site}->{history}->{$generation}}) {
my $event_num = $site_to_outgroups{$mutated_site}->{history}->{$generation}->{$event};
if($event =~ /(\d+)\-([\>\w+]+)/) {
my $this_mutation = $2;
$mutations_out .= "$2\,";
$generations_out .= "$1\,";
$taxa_out .= $event_num . ',';
my @two_states = split(/>/, $this_mutation);
my $state1 = $two_states[0];
my $state2 = $two_states[1];
# Could be recurrent/parallel
if ($prev_change_out{$this_mutation}) {
$recurrent_mutation_out++;
}
$prev_change_out{$this_mutation}++;
# Could be back mutation
if ($prev_state_1_out{$state2}) {
$back_mutation_out++;
}
$prev_state_1_out{$state1}++;
$prev_state_2_out{$state2}++;
}
}
}
chop($mutations_out);
chop($generations_out);
chop($taxa_out);
if ($mutations_out ne '' || $generations_out ne '' || $taxa_out ne '') {
$out_line .= "MUTATIONS_OG=$mutations_out\;GENERATIONS_OG=$generations_out\;TAXA_OG=$taxa_out\;";
}
}
##################################################################################
# INGROUP (SNP) FLAGS
if ($arbitrary_REF == 1) {
$out_line .= "ARBITRARY_REF\;";
}
if ($hits > 1) {
$out_line .= "MULTIHIT\;";
}
if ($num_alleles > 2) {
$out_line .= "MULTIALLELIC\;";
}
if ($back_mutation > 0) {
$out_line .= "BACK_MUTATION\;";
}
if ($recurrent_mutation > 0) {
$out_line .= "RECURRENT_MUTATION\;";
}
if ($AF eq '1') {
if ($REF eq $AA) {
$out_line .= "INVARIANT_ANCESTRAL\;";
} else {
$out_line .= "INVARIANT_DERIVED\;";
}
}
if ($REF ne $AA) {
my $match_AA = 0;
foreach (@all_nts_present) {
if ($_ eq $AA) {
$match_AA = 1;
}
}
# Unless we have a match to the ancestral allele SOMEWHERE
unless ($match_AA == 1) {
$out_line .= "NO_ANCESTRAL\;";
}
}
##################################################################################
# OUTGROUP (SUB) FLAGS
if ($outgroups) {
# Define REF as the major (consensus) allele
my $REF_OG = '';
my $REF_OG_count = 0;
foreach my $nt (@nts) {
if ($site_to_outgroups{$mutated_site}->{$nt} > $REF_OG_count) {
$REF_OG = $nt;
$REF_OG_count = $site_to_outgroups{$mutated_site}->{$nt};
}
}
my $num_alleles_out = 0;
my $num_seqs_out = 0;
my @all_nts_present_out;
my $outgroup_alleles = '';
my $outgroup_counts = '';
foreach my $nt (@nts) {
if ($site_to_outgroups{$mutated_site}->{$nt} > 0) { # here, possible that back mutation eliminates variation
push(@all_nts_present_out, $nt);
$num_alleles_out++;