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snpgenie_between_group_processor.pl
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snpgenie_between_group_processor.pl
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#! /usr/bin/perl
#AMNH-HUXLEY-PBS is #! /usr/local/software/PERL/perl-5.26.0/bin/perl
#NYU-PRINCE-SLURM is #! /share/apps/perl/5.24.0/intel/bin/perl
#PC is #! /usr/bin/perl
# POST-PROCESSING for PhyloGeneious.
# PROGRAM: Recalculate whole-product and sliding window dN/dS values FOR BETWEEN-GROUP
# ANALYSES using a given window and step size for all families/products, and criteria
# for number of taxa present in the alignment overall (total) or in each group (group).
# REQUIRES: the existence of "between_group_codon_results.txt" files, produced from an
# initial run of SNPGenie, in all family directories (i.e.,
# $OID_USER_DIR/data/*/between_group_codon_results.txt).
#########################################################################################
# CALL FORMAT:
#########################################################################################
# snpgenie_between_group_processor.pl <input_file> <codon_min_taxa_total> <codon_min_taxa_group> <sw_size> <sw_step_size> <num_bootstraps> <procs_per_node>
#########################################################################################
#########################################################################################
# EXAMPLE CALLS:
#########################################################################################
# snpgenie_between_group_processor.pl --between_group_codon_file=between_group_codon_results.txt --sliding_window_size=8 --sliding_window_step=1 --num_bootstraps=1000 --procs_per_node=4
# snpgenie_between_group_processor.pl --between_group_codon_file=between_group_codon_results.txt --sliding_window_size=1 --sliding_window_step=1 --num_bootstraps=1000 --procs_per_node=1 --codon_min_taxa_total=5 --codon_min_taxa_group=2
#########################################################################################
# OUTPUT:
# (1) between_group_sw_<#>codons_results.txt
# If the filtering criteria (codon_min_taxa_total and/or codon_min_taxa_group) are
# NOT met, then the columns N_sites, S_sites, N_diffs, S_diffs dN>total_dS,
# SE(dN-dS), and Z_value will be populated with NA values. REPLACES file by the same
# name if it already exists.
# (2) between_group_results_filtered.txt
# Identical to the original between_group_results.txt, but with values calculated
# excluding codons which do not meet filtering criteria. REPLACES file by the same
# name if it already exists.
# Copyright (C) 2017 Chase W. Nelson
#########################################################################################
## LICENSE
## This program is free software: you can redistribute it and/or modify
## it under the terms of the GNU General Public License as published by
## the Free Software Foundation, either version 3 of the License, or
## (at your option) any later version.
##
## This program is distributed in the hope that it will be useful,
## but WITHOUT ANY WARRANTY; without even the implied warranty of
## MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the
## GNU General Public License for more details.
##
## You should have received a copy of the GNU General Public License
## along with this program. If not, see <http://www.gnu.org/licenses/>.
#########################################################################################
# DATE CREATED: November 9, 2017
# AUTHOR: Chase W. Nelson
# CONTACT1: [email protected]
# CONTACT2: [email protected]
# AFFILIATION1: Sackler Institute for Comparative Genomics, American Museum of Natural
# History, New York, NY 10024, USA
# AFFILIATION2: Special Volunteer, Division of Cancer Epidemiology & Genetics, National
# Cancer Institute, National Institutes of Health, Rockville, MD 20850, USA
# AFFILIATION3: BigPlant Consortium, Center for Genomics and Systems Biology, New York
# University, New York, NY 10003, USA
# CITATION1: SNPGenie, https://github.com/chasewnelson/snpgenie
# CITATION2: Nelson CW, Moncla LH, Hughes AL (2015) SNPGenie: estimating evolutionary
# parameters to detect natural selection using pooled next-generation sequencing data.
# Bioinformatics 31(22):3709-11, doi: 10.1093/bioinformatics/btv449.
use strict;
#use warnings;
use Data::Dumper;
use Getopt::Long;
use List::Util qw(max);
use Parallel::ForkManager;
#########################################################################################
# INITIALIZE VARIABLES (all optional and/or defaulted)
my $between_group_codon_file;
my $sliding_window_size;
my $sliding_window_step;
my $codon_min_taxa_total;
my $codon_min_taxa_group;
my $num_bootstraps;
my $procs_per_node;
# Get user input, if given. If a Boolean argument is passed, its value is 1; else undef
GetOptions( "between_group_codon_file=s" => \$between_group_codon_file,
"sliding_window_size=i" => \$sliding_window_size,
"sliding_window_step=i" => \$sliding_window_step,
"codon_min_taxa_total=i" => \$codon_min_taxa_total,
"codon_min_taxa_group=i" => \$codon_min_taxa_group,
"num_bootstraps=i" => \$num_bootstraps,
"procs_per_node:i" => \$procs_per_node)
or die "\n### WARNING: Error in command line arguments. SNPGenie terminated.\n\n";
# Get the time
my $time1 = time;
my $local_time1 = localtime;
print "\n#########################################################################################";
print "\n##### SNPGenie filtering, sliding windows, and bootstrapping initiated at local time $local_time1\n";
print "#########################################################################################";
print "\n";
# Test and/or reset OPTIONS given the user's INPUT and RECORD PARAMETERS
if(! $between_group_codon_file) {
die "\n### WARNING: The --between_group_codon_file option must be provided\n".
"### SNPGenie terminated.\n\n";
}
# Test and/or reset OPTIONS given the user's INPUT and RECORD PARAMETERS
if(! $sliding_window_size) {
if($sliding_window_size != 0) { # Called as a flag, but given no value
$sliding_window_size = 10; # default behavior: 10-mer peptide
}
} elsif($sliding_window_size < 1) {
die "\n### WARNING: The --sliding_window_size option must be an integer ≥1\n".
"### SNPGenie terminated.\n\n";
}
if(! $sliding_window_step) {
if($sliding_window_step != 0) { # Called as a flag, but given no value
$sliding_window_step = 1; # default behavior: one codon
}
} elsif($sliding_window_step < 1) {
die "\n### WARNING: The --sliding_window_step option must be an integer ≥1\n".
"### SNPGenie terminated.\n\n";
}
if(! $codon_min_taxa_total) { # null or 0
$codon_min_taxa_total = 0; # default behavior
} elsif(! $codon_min_taxa_total >= 1) {
die "\n### WARNING: The --codon_min_taxa_total option must ≥1\n".
"### Script terminated.\n\n";
}
if(! $codon_min_taxa_group) { # null or 0
$codon_min_taxa_group = 0; # default behavior
} elsif(! $codon_min_taxa_group >= 1) {
die "\n### WARNING: The --codon_min_taxa_group option must ≥1\n".
"### Script terminated.\n\n";
}
if(! $num_bootstraps) {
$num_bootstraps = 0; # DEFAULT is 0
print "### BOOTSTRAP VALUE NOT GIVEN. DEFAULT IS NO BOOTSTRAPPING.\n" .
"### (You sure you don't want our statistics?)\n\n";
} elsif($num_bootstraps < 100) {
die "\n### WARNING: The --num_bootstraps option must be an integer ≥100\n".
"### SNPGenie terminated.\n\n";
}
if(! $procs_per_node) {
$procs_per_node = 1; # DEFAULT is 1
print "### PROCS PER NODE VALUE NOT GIVEN. DEFAULT USED: JUST 1.\n" .
"### (You sure you don't want our parallelization?)\n\n";
} elsif($procs_per_node < 1) {
die "\n### WARNING: The --procs_per_node option must be an integer ≥1\n".
"### SNPGenie terminated.\n\n";
}
#STDOUT->autoflush(1);
#########################################################################################
# Input and output file names; same for every family directory
#my $between_group_codon_file = 'between_group_codon_results.txt';
my $between_group_sw_output_file = 'between_group_sw_' . $sliding_window_size . 'codons_results.txt';
my $between_group_product_results_file = 'between_group_product_results_filtered.txt'; # later, looks like between\_group\_results\_filtered\.txt
#########################################################################################
# BEGIN PROCESSING
print "\n#########################################################################################";
print "\n##### Working on file $between_group_codon_file\n";
print "#########################################################################################";
print "\n";
if(-f "$between_group_codon_file") { # IF THE INPUT FILE EXISTS
##################################################################################
# READY THE OUTPUT FILE FOR SLIDING WINDOWS
if(-f "$between_group_sw_output_file") { # file already exists
warn "\n### WARNING: $between_group_sw_output_file already exists in this directory.\n".
"### REMOVING AND REPLACING using current criteria.\n";
unlink $between_group_sw_output_file;
}
open(OUTFILE_BETWEEN_SW,">$between_group_sw_output_file");
# print OUTFILE_BETWEEN_SW "analysis\tfamily\tproduct\tgroup_1\tgroup_2\twindow\tfirst_codon\tlast_codon\t". ###???
print OUTFILE_BETWEEN_SW "analysis\tproduct\tgroup_1\tgroup_2\twindow\tfirst_codon\tlast_codon\t". ###???
"num_defined_codons_g1\tnum_defined_codons_g2\tmean_num_defined_codons\tmin_num_defined_codons\t".
"N_sites\tS_sites\t".
"N_diffs\tS_diffs\t".
"dN\tdS\t";
if($num_bootstraps > 1) {
print OUTFILE_BETWEEN_SW "SE_dN\tSE_dS\t";
}
print OUTFILE_BETWEEN_SW "dN_minus_dS\tdN_over_dS";
if($num_bootstraps > 1) {
print OUTFILE_BETWEEN_SW "\tSE_dN_minus_dS\tZ_value\tsignificance\n";
} else {
print OUTFILE_BETWEEN_SW "\n";
}
close OUTFILE_BETWEEN_SW;
##################################################################################
# READY THE OUTPUT FILE FOR PRODUCT
if(-f "$between_group_product_results_file") { # file already exists
warn "\n### WARNING: $between_group_product_results_file already exists in this directory.\n".
"### REMOVING AND REPLACING using current criteria.\n";
unlink "$between_group_product_results_file";
}
open(BETWEEN_OUTFILE,">>$between_group_product_results_file");
# print BETWEEN_OUTFILE "analysis\tfamily\tproduct\tgroup_1\tgroup_2\tN_sites\tS_sites\tN_diffs\tS_diffs\t". ###???
print BETWEEN_OUTFILE "analysis\tproduct\tgroup_1\tgroup_2\tN_sites\tS_sites\tN_diffs\tS_diffs\t". ###???
"dN\tdS\tJC_dN\tJC_dS\t";
if($num_bootstraps > 1) {
print BETWEEN_OUTFILE "SE_dN\tSE_dS\tSE_JC_dN\tSE_JC_dS\t";
}
print BETWEEN_OUTFILE "dN_minus_dS\tdNdS\tJC_dN_minus_dS\tJC_dNdS";
if($num_bootstraps > 1) {
print BETWEEN_OUTFILE "\tSE_dN_minus_dS\tZ_value\tsignificance\t" .
"SE_JC_dN_minus_dS\tJC_Z_value\tJC_significance\n";
} else {
print BETWEEN_OUTFILE "\n";
}
close BETWEEN_OUTFILE;
##################################################################################
# READY THE VARIABLES FOR THE WHOLE PARTITION
# my %site_data_hh;
# # $site_data_hh{<site>}->{<N_diffs>};
# # $site_data_hh{<site>}->{<S_diffs>};
# # $site_data_hh{<site>}->{<N_sites>};
# # $site_data_hh{<site>}->{<S_sites>};
# # $site_data_hh{<site>}->{<group_1_#_taxa>};
# # $site_data_hh{<site>}->{<group_2_#_taxa>};
##################################################################################
# STORE BETWEEN-GROUP CODON DATA
# Get header names
my @header_names_arr = &get_header_names($between_group_codon_file,$between_group_codon_file);
#print "@header_names_arr";
my %header_indices;
# Determine the index of each column
for (my $i = 0; $i < scalar(@header_names_arr); $i++) {
my $curr_header = $header_names_arr[$i];
$header_indices{$curr_header} = $i;
}
# Make sure we got all the expected headers
# my @required_between_group_headers = qw/analysis family product group_1 group_2 codon variability num_defined_codons_g1 num_defined_codons_g2 comparisons N_sites S_sites N_diffs S_diffs/; ###???
my @required_between_group_headers = qw/analysis product group_1 group_2 codon variability num_defined_codons_g1 num_defined_codons_g2 comparisons N_sites S_sites N_diffs S_diffs/; ###???
foreach(@required_between_group_headers) {
unless(exists $header_indices{$_}) {
die "### DIE: the header name $_ is not present in the between-group codon results file.\n\n";
}
}
##################################################################################
# STORE ALL FAMILY BETWEEN-GROUP CODON DATA
my %between_group_codon_data_hh;
my %between_group_a_p_groups;
##################################################################################
# STORE overall product values
my %between_group_a_p_sums;
### my $between_product_N_sites_sum = 0;
## my $between_product_S_sites_sum = 0;
### my $between_product_N_diffs_sum = 0;
## my $between_product_S_diffs_sum = 0;
##################################################################################
# LOOP CODONS TO STORE DATA IN %between_group_codon_data_hh
open(IN_BETWEEN_GROUP_CODON_FILE, "$between_group_codon_file") or die "\nCould not open the file $between_group_codon_file - $!\n\n";
my $line = 0;
while(<IN_BETWEEN_GROUP_CODON_FILE>) { # record
if($line == 0) {
$line++;
} else {
chomp;
my @line_arr = split(/\t/,$_,-1);
# Store data here
my $analysis = $line_arr[$header_indices{'analysis'}];
# my $analysis = "only_one";
# my $family = $line_arr[$header_indices{'family'}];
# my $product = $line_arr[$header_indices{'product'}];
my $product = $line_arr[$header_indices{'product'}];
my $codon = $line_arr[$header_indices{'codon'}];
$codon =~ s/codon_//; # in case it's the old format
$codon =~ s/\s//g; # in case it's the old format
# Store the names of the groups in this pair
my @curr_groups = sort ($line_arr[$header_indices{'group_1'}], $line_arr[$header_indices{'group_2'}]);
my $this_group_1 = $curr_groups[0];
my $this_group_2 = $curr_groups[1];
$between_group_codon_data_hh{$analysis}->{$this_group_1}->{$this_group_2}->{$product}->{$codon}->{variability} = $line_arr[$header_indices{'variability'}];
$between_group_codon_data_hh{$analysis}->{$this_group_1}->{$this_group_2}->{$product}->{$codon}->{num_defined_codons_g1} = $line_arr[$header_indices{'num_defined_codons_g1'}];
$between_group_codon_data_hh{$analysis}->{$this_group_1}->{$this_group_2}->{$product}->{$codon}->{num_defined_codons_g2} = $line_arr[$header_indices{'num_defined_codons_g2'}];
$between_group_codon_data_hh{$analysis}->{$this_group_1}->{$this_group_2}->{$product}->{$codon}->{comparisons} = $line_arr[$header_indices{'comparisons'}];
$between_group_codon_data_hh{$analysis}->{$this_group_1}->{$this_group_2}->{$product}->{$codon}->{N_sites} = $line_arr[$header_indices{'N_sites'}];
$between_group_codon_data_hh{$analysis}->{$this_group_1}->{$this_group_2}->{$product}->{$codon}->{S_sites} = $line_arr[$header_indices{'S_sites'}];
$between_group_codon_data_hh{$analysis}->{$this_group_1}->{$this_group_2}->{$product}->{$codon}->{N_diffs} = $line_arr[$header_indices{'N_diffs'}];
$between_group_codon_data_hh{$analysis}->{$this_group_1}->{$this_group_2}->{$product}->{$codon}->{S_diffs} = $line_arr[$header_indices{'S_diffs'}];
# print "\n\nVariability: " . $between_group_codon_data_hh{$analysis}->{$family}->{$product}->{$codon}->{variability} . "\n\n";
# print "\n\nN_diffs: " . $between_group_codon_data_hh{$analysis}->{$family}->{$product}->{$codon}->{N_diffs} . "\n\n";
# print "\nSTORING:\nanalysis: $analysis\nfamily: $family\nproduct: $product\ncodon: $codon\nNsites: ".
# $line_arr[$header_indices{'N_sites'}] . "\n\n";
# THERE ARE MORE THAN 2.....
# Store group names here
# $between_group_a_p_groups{$analysis}->{$product}->{group_1} = $line_arr[$header_indices{'group_1'}];
# $between_group_a_p_groups{$analysis}->{$product}->{group_2} = $line_arr[$header_indices{'group_2'}];
# No longer needed because used as keys in the hh
# Sum product values here, NONFILTERED
$between_group_a_p_sums{$analysis}->{$this_group_1}->{$this_group_2}->{$product}->{N_sites} += $line_arr[$header_indices{'N_sites'}];
$between_group_a_p_sums{$analysis}->{$this_group_1}->{$this_group_2}->{$product}->{S_sites} += $line_arr[$header_indices{'S_sites'}];
$between_group_a_p_sums{$analysis}->{$this_group_1}->{$this_group_2}->{$product}->{N_diffs} += $line_arr[$header_indices{'N_diffs'}];
$between_group_a_p_sums{$analysis}->{$this_group_1}->{$this_group_2}->{$product}->{S_diffs} += $line_arr[$header_indices{'S_diffs'}];
}
}
close IN_BETWEEN_GROUP_CODON_FILE;
#### THIS DIFFERS FOR EACH BETWEEN-GROUP ANALYSIS, SO MUST BE RE-CALCULATED FOR EACH
# my $between_product_dS;
# if($between_product_S_sites_sum_filtered > 0) {
# $between_product_dS = $between_product_S_diffs_sum_filtered / $between_product_S_sites_sum_filtered;
# } else {
# $between_product_dS = '*';
# }
##################################################################################
# LOOP THROUGH EACH ANALYSIS, FAMILY, AND PARTITION FOR SLIDING WINDOW ANALYSIS
foreach my $this_analysis (sort keys %between_group_codon_data_hh) {
foreach my $group_name_i (sort keys %{$between_group_codon_data_hh{$this_analysis}}) {
foreach my $group_name_j (sort keys %{$between_group_codon_data_hh{$this_analysis}->{$group_name_i}}) {
foreach my $this_product (sort {$a <=> $b} keys %{$between_group_codon_data_hh{$this_analysis}->{$group_name_i}->{$group_name_j}}) {
# Get group names
# my $group_name_i = $between_group_a_p_groups{$this_analysis}->{$this_product}->{group_1};
# my $group_name_j = $between_group_a_p_groups{$this_analysis}->{$this_product}->{group_2};
# print "\nTEST RETRIEVAL:\nanalysis: $this_analysis\nfamily: $this_family\nproduct: $this_product\n\n";
#############################################################
# SLIDING WINDOW WITH BOOTSTRAPS
# Generate and store sliding window and bootstrap analyses
my %sliding_window_results_hh; # Will store first_codon->N_diffs_window/S_diffs_window/...
# my $sw_file_name = "product$product_id\_sw_" . $sliding_window_size . "codons\_results.txt";
#
# if (-e "$sw_file_name") {
# die "\n\n### WARNING:\n## The file $sw_file_name already exists.\n## Please ".
# "rename or move this file so a new one ".
# "can be created.\n\n";
# }
my @codons = keys (%{$between_group_codon_data_hh{$this_analysis}->{$group_name_i}->{$group_name_j}->{$this_product}});
my @sorted_codons = sort {$a <=> $b} (@codons);
my $num_sorted_codons = scalar @sorted_codons;
# print "\n\ncodons: @sorted_codons\n\n";
if($sliding_window_size > $num_sorted_codons) {
die "\n\n### WARNING:\n## For product $this_product\,\n## there are ".
"not enough codons to perform a sliding window of $sliding_window_size.\n\n";
}
######################################################################
# STEP THROUGH CODONS AND FILTER VALUES
my $between_product_N_sites_sum_filtered = 0;
my $between_product_S_sites_sum_filtered = 0;
my $between_product_N_diffs_sum_filtered = 0;
my $between_product_S_diffs_sum_filtered = 0;
for (my $codon_index = 0; $codon_index <= $num_sorted_codons; $codon_index++) {
my $codon_num = $codon_index + 1;
my $num_defined_codons_g1 = $between_group_codon_data_hh{$this_analysis}->{$group_name_i}->{$group_name_j}->{$this_product}->{$codon_num}->{'num_defined_codons_g1'};
my $num_defined_codons_g2 = $between_group_codon_data_hh{$this_analysis}->{$group_name_i}->{$group_name_j}->{$this_product}->{$codon_num}->{'num_defined_codons_g2'};
# print "\nnum_defined_codons_g1 is $num_defined_codons_g1\n";
if($codon_min_taxa_total > 0 && $codon_min_taxa_group > 0) { # BOTH criteria have been specified
my $taxa_total = $num_defined_codons_g1 + $num_defined_codons_g2;
my $taxa_group_min = $num_defined_codons_g1;
if($num_defined_codons_g2 < $taxa_group_min) {
$taxa_group_min = $num_defined_codons_g2;
}
if($taxa_total >= $codon_min_taxa_total && $taxa_group_min >= $codon_min_taxa_group) { # see if criteria are met by this codon
$between_product_N_sites_sum_filtered += $between_group_codon_data_hh{$this_analysis}->{$group_name_i}->{$group_name_j}->{$this_product}->{$codon_num}->{N_sites};
$between_product_S_sites_sum_filtered += $between_group_codon_data_hh{$this_analysis}->{$group_name_i}->{$group_name_j}->{$this_product}->{$codon_num}->{S_sites};
$between_product_N_diffs_sum_filtered += $between_group_codon_data_hh{$this_analysis}->{$group_name_i}->{$group_name_j}->{$this_product}->{$codon_num}->{N_diffs};
$between_product_S_diffs_sum_filtered += $between_group_codon_data_hh{$this_analysis}->{$group_name_i}->{$group_name_j}->{$this_product}->{$codon_num}->{S_diffs};
}
} elsif($codon_min_taxa_total > 0) { # only TOTAL min has been specified
my $taxa_total = $num_defined_codons_g1 + $num_defined_codons_g2;
if($taxa_total >= $codon_min_taxa_total) { # see if criterion is met by this codon
$between_product_N_sites_sum_filtered += $between_group_codon_data_hh{$this_analysis}->{$group_name_i}->{$group_name_j}->{$this_product}->{$codon_num}->{N_sites};
$between_product_S_sites_sum_filtered += $between_group_codon_data_hh{$this_analysis}->{$group_name_i}->{$group_name_j}->{$this_product}->{$codon_num}->{S_sites};
$between_product_N_diffs_sum_filtered += $between_group_codon_data_hh{$this_analysis}->{$group_name_i}->{$group_name_j}->{$this_product}->{$codon_num}->{N_diffs};
$between_product_S_diffs_sum_filtered += $between_group_codon_data_hh{$this_analysis}->{$group_name_i}->{$group_name_j}->{$this_product}->{$codon_num}->{S_diffs};
}
} elsif($codon_min_taxa_group > 0) { # only GROUP min has been specified
my $taxa_group_min = $num_defined_codons_g1;
if($num_defined_codons_g2 < $taxa_group_min) {
$taxa_group_min = $num_defined_codons_g2;
}
if($taxa_group_min >= $codon_min_taxa_group) { # see if criterion is met by this codon
$between_product_N_sites_sum_filtered += $between_group_codon_data_hh{$this_analysis}->{$group_name_i}->{$group_name_j}->{$this_product}->{$codon_num}->{N_sites};
$between_product_S_sites_sum_filtered += $between_group_codon_data_hh{$this_analysis}->{$group_name_i}->{$group_name_j}->{$this_product}->{$codon_num}->{S_sites};
$between_product_N_diffs_sum_filtered += $between_group_codon_data_hh{$this_analysis}->{$group_name_i}->{$group_name_j}->{$this_product}->{$codon_num}->{N_diffs};
$between_product_S_diffs_sum_filtered += $between_group_codon_data_hh{$this_analysis}->{$group_name_i}->{$group_name_j}->{$this_product}->{$codon_num}->{S_diffs};
}
} else { # BOTH CRITERIA 0 OR UNSPECIFIED
$between_product_N_sites_sum_filtered += $between_group_codon_data_hh{$this_analysis}->{$group_name_i}->{$group_name_j}->{$this_product}->{$codon_num}->{N_sites};
$between_product_S_sites_sum_filtered += $between_group_codon_data_hh{$this_analysis}->{$group_name_i}->{$group_name_j}->{$this_product}->{$codon_num}->{S_sites};
$between_product_N_diffs_sum_filtered += $between_group_codon_data_hh{$this_analysis}->{$group_name_i}->{$group_name_j}->{$this_product}->{$codon_num}->{N_diffs};
$between_product_S_diffs_sum_filtered += $between_group_codon_data_hh{$this_analysis}->{$group_name_i}->{$group_name_j}->{$this_product}->{$codon_num}->{S_diffs};
}
}
my $between_product_dS_filtered;
if($between_product_S_sites_sum_filtered > 0) {
$between_product_dS_filtered = $between_product_S_diffs_sum_filtered / $between_product_S_sites_sum_filtered;
} else {
$between_product_dS_filtered = '*';
}
my $between_product_dN_filtered;
if($between_product_N_sites_sum_filtered > 0) {
$between_product_dN_filtered = $between_product_N_diffs_sum_filtered / $between_product_N_sites_sum_filtered;
} else {
$between_product_dN_filtered = '*';
}
# Jukes-Cantor Correction
my $between_JC_dN = -3/4 * log(1 - ((4/3) * $between_product_dN_filtered));
my $between_JC_dS = -3/4 * log(1 - ((4/3) * $between_product_dS_filtered));
# print "\nANALYSIS $this_analysis FAMILY $this_family PARTITION $this_product\n".
# "filtered dN = $between_product_dN_filtered filtered dS = $between_product_dS_filtered\n";
######################################################################
# STEP THROUGH WINDOWS, IGNORE CODONS THAT DON'T FIT CRITERIA
# FOR EACH WINDOW
my $window_num = 0;
my $first_print = 0;
# print "\nProcessing sliding windows...\n";
# CODON_INDEX must begin one before the smallest actual codon number... don't assume it's 1
for (my $codon_index = 0; $codon_index <= ($num_sorted_codons - $sliding_window_size); $codon_index += $sliding_window_step) {
$window_num++;
my $first_codon = $codon_index + $sorted_codons[0];
my $last_codon = $first_codon + $sliding_window_size - 1;
# print "#### PROCESSING WINDOW $window_num\, codons $first_codon\-$last_codon\... \n";
if ($last_codon <= ($num_sorted_codons + $sorted_codons[0])) { # as long as we're in range
my $window_N_diffs;
my $window_S_diffs;
my $window_N_sites;
my $window_S_sites;
for (my $i = $first_codon; $i <= $last_codon; $i++) {
# print "\n\nTo codon $i, we add: " . $between_group_codon_data_hh{$this_analysis}->{$group_name_i}->{$group_name_j}->{$this_product}->{$i}->{N_diffs} . "\n\n";
# print "\nTEST RETRIEVAL:\nanalysis: $this_analysis\nfamily: $this_family\nproduct: $this_product\n\n";
my $num_defined_codons_g1 = $between_group_codon_data_hh{$this_analysis}->{$group_name_i}->{$group_name_j}->{$this_product}->{$i}->{'num_defined_codons_g1'};
my $num_defined_codons_g2 = $between_group_codon_data_hh{$this_analysis}->{$group_name_i}->{$group_name_j}->{$this_product}->{$i}->{'num_defined_codons_g2'};
if($codon_min_taxa_total > 0 && $codon_min_taxa_group > 0) { # BOTH criteria have been specified
my $taxa_total = $num_defined_codons_g1 + $num_defined_codons_g2;
my $taxa_group_min = $num_defined_codons_g1;
if($num_defined_codons_g2 < $taxa_group_min) {
$taxa_group_min = $num_defined_codons_g2;
}
if($taxa_total >= $codon_min_taxa_total && $taxa_group_min >= $codon_min_taxa_group) { # see if criteria are met by this codon
$window_N_diffs += $between_group_codon_data_hh{$this_analysis}->{$group_name_i}->{$group_name_j}->{$this_product}->{$i}->{N_diffs};
$window_S_diffs += $between_group_codon_data_hh{$this_analysis}->{$group_name_i}->{$group_name_j}->{$this_product}->{$i}->{S_diffs};
$window_N_sites += $between_group_codon_data_hh{$this_analysis}->{$group_name_i}->{$group_name_j}->{$this_product}->{$i}->{N_sites};
$window_S_sites += $between_group_codon_data_hh{$this_analysis}->{$group_name_i}->{$group_name_j}->{$this_product}->{$i}->{S_sites};
}
} elsif($codon_min_taxa_total > 0) { # only TOTAL min has been specified
my $taxa_total = $num_defined_codons_g1 + $num_defined_codons_g2;
if($taxa_total >= $codon_min_taxa_total) { # see if criterion is met by this codon
$window_N_diffs += $between_group_codon_data_hh{$this_analysis}->{$group_name_i}->{$group_name_j}->{$this_product}->{$i}->{N_diffs};
$window_S_diffs += $between_group_codon_data_hh{$this_analysis}->{$group_name_i}->{$group_name_j}->{$this_product}->{$i}->{S_diffs};
$window_N_sites += $between_group_codon_data_hh{$this_analysis}->{$group_name_i}->{$group_name_j}->{$this_product}->{$i}->{N_sites};
$window_S_sites += $between_group_codon_data_hh{$this_analysis}->{$group_name_i}->{$group_name_j}->{$this_product}->{$i}->{S_sites};
}
} elsif($codon_min_taxa_group > 0) { # only GROUP min has been specified
my $taxa_group_min = $num_defined_codons_g1;
if($num_defined_codons_g2 < $taxa_group_min) {
$taxa_group_min = $num_defined_codons_g2;
}
if($taxa_group_min >= $codon_min_taxa_group) { # see if criterion is met by this codon
$window_N_diffs += $between_group_codon_data_hh{$this_analysis}->{$group_name_i}->{$group_name_j}->{$this_product}->{$i}->{N_diffs};
$window_S_diffs += $between_group_codon_data_hh{$this_analysis}->{$group_name_i}->{$group_name_j}->{$this_product}->{$i}->{S_diffs};
$window_N_sites += $between_group_codon_data_hh{$this_analysis}->{$group_name_i}->{$group_name_j}->{$this_product}->{$i}->{N_sites};
$window_S_sites += $between_group_codon_data_hh{$this_analysis}->{$group_name_i}->{$group_name_j}->{$this_product}->{$i}->{S_sites};
}
} else { # BOTH CRITERIA 0 OR UNSPECIFIED
$window_N_diffs += $between_group_codon_data_hh{$this_analysis}->{$group_name_i}->{$group_name_j}->{$this_product}->{$i}->{N_diffs};
$window_S_diffs += $between_group_codon_data_hh{$this_analysis}->{$group_name_i}->{$group_name_j}->{$this_product}->{$i}->{S_diffs};
$window_N_sites += $between_group_codon_data_hh{$this_analysis}->{$group_name_i}->{$group_name_j}->{$this_product}->{$i}->{N_sites};
$window_S_sites += $between_group_codon_data_hh{$this_analysis}->{$group_name_i}->{$group_name_j}->{$this_product}->{$i}->{S_sites};
}
}
my $window_dN;
if ($window_N_sites > 0) {
$window_dN = ($window_N_diffs / $window_N_sites);
} else {
$window_dN = '*';
}
my $window_dS;
if ($window_S_sites > 0) {
$window_dS = ($window_S_diffs / $window_S_sites);
} else {
$window_dS = '*';
}
my $window_w;
if ($window_dS > 0 && $window_dS ne '*') {
$window_w = ($window_dN / $window_dS);
} else {
$window_w = '*';
}
my $window_dN_minus_dS;
if($window_dN >= 0 && $window_dS >= 0) {
$window_dN_minus_dS = $window_dN - $window_dS;
}
# print "\n\n";
#################################################################################
# BOOTSTRAP the WINDOW
my $window_SE_dN_minus_dS = 0;
my $window_boot_Z = 'NA';
my $window_SE_dN;
my $window_SE_dS;
if($num_bootstraps > 1) {
# MAKE BOOTSTRAP FILE
#print "num_codons_in_product: $num_codons_in_product\n";
my @sim_N_sites_arr;
my @sim_S_sites_arr;
my @sim_N_diffs_arr;
my @sim_S_diffs_arr;
for(my $bootstrap_num = 1; $bootstrap_num <= $num_bootstraps; $bootstrap_num++) {
srand();
# New bootstrap run, of $num_bootstraps
#if($this_family == 460) {
# print "\nPartition $this_product \| BOOTSTRAP REPLICATE $bootstrap_num\n";
#}
my $sim_N_sites_sum = 0;
my $sim_S_sites_sum = 0;
my $sim_N_diffs_sum = 0;
my $sim_S_diffs_sum = 0;
# METHOD
# (1) loop through $num_codons_in_product
# (2) for each, select a random codon position's results: Nd, Sd, Ns, Ss.
# where are the aforementioned stored? As before:
# $between_group_codon_data_hh{$this_analysis}->{$group_name_i}->{$group_name_j}->{$this_product}->{$codon_num}->{N_diffs}, etc.
# SAMPLE codon sites, up to the actual number of codons in window
for(my $window_codon_index = 0; $window_codon_index < $sliding_window_size; $window_codon_index++) {
# Choose a random codon between this window $first_codon and $last_codon
my $random_codon_num = int(rand($last_codon - $first_codon + 1)) + $first_codon;
my $num_defined_codons_g1 = $between_group_codon_data_hh{$this_analysis}->{$group_name_i}->{$group_name_j}->{$this_product}->{$random_codon_num}->{'num_defined_codons_g1'};
my $num_defined_codons_g2 = $between_group_codon_data_hh{$this_analysis}->{$group_name_i}->{$group_name_j}->{$this_product}->{$random_codon_num}->{'num_defined_codons_g2'};
if($codon_min_taxa_total > 0 && $codon_min_taxa_group > 0) { # BOTH criteria have been specified
my $taxa_total = $num_defined_codons_g1 + $num_defined_codons_g2;
my $taxa_group_min = $num_defined_codons_g1;
if($num_defined_codons_g2 < $taxa_group_min) {
$taxa_group_min = $num_defined_codons_g2;
}
if($taxa_total >= $codon_min_taxa_total && $taxa_group_min >= $codon_min_taxa_group) { # see if criteria are met by this codon
$sim_N_diffs_sum += $between_group_codon_data_hh{$this_analysis}->{$group_name_i}->{$group_name_j}->{$this_product}->{$random_codon_num}->{N_diffs};
$sim_S_diffs_sum += $between_group_codon_data_hh{$this_analysis}->{$group_name_i}->{$group_name_j}->{$this_product}->{$random_codon_num}->{S_diffs};
$sim_N_sites_sum += $between_group_codon_data_hh{$this_analysis}->{$group_name_i}->{$group_name_j}->{$this_product}->{$random_codon_num}->{N_sites};
$sim_S_sites_sum += $between_group_codon_data_hh{$this_analysis}->{$group_name_i}->{$group_name_j}->{$this_product}->{$random_codon_num}->{S_sites};
}
} elsif($codon_min_taxa_total > 0) { # only TOTAL min has been specified
my $taxa_total = $num_defined_codons_g1 + $num_defined_codons_g2;
if($taxa_total >= $codon_min_taxa_total) { # see if criterion is met by this codon
$sim_N_diffs_sum += $between_group_codon_data_hh{$this_analysis}->{$group_name_i}->{$group_name_j}->{$this_product}->{$random_codon_num}->{N_diffs};
$sim_S_diffs_sum += $between_group_codon_data_hh{$this_analysis}->{$group_name_i}->{$group_name_j}->{$this_product}->{$random_codon_num}->{S_diffs};
$sim_N_sites_sum += $between_group_codon_data_hh{$this_analysis}->{$group_name_i}->{$group_name_j}->{$this_product}->{$random_codon_num}->{N_sites};
$sim_S_sites_sum += $between_group_codon_data_hh{$this_analysis}->{$group_name_i}->{$group_name_j}->{$this_product}->{$random_codon_num}->{S_sites};
}
} elsif($codon_min_taxa_group > 0) { # only GROUP min has been specified
my $taxa_group_min = $num_defined_codons_g1;
if($num_defined_codons_g2 < $taxa_group_min) {
$taxa_group_min = $num_defined_codons_g2;
}
if($taxa_group_min >= $codon_min_taxa_group) { # see if criterion is met by this codon
$sim_N_diffs_sum += $between_group_codon_data_hh{$this_analysis}->{$group_name_i}->{$group_name_j}->{$this_product}->{$random_codon_num}->{N_diffs};
$sim_S_diffs_sum += $between_group_codon_data_hh{$this_analysis}->{$group_name_i}->{$group_name_j}->{$this_product}->{$random_codon_num}->{S_diffs};
$sim_N_sites_sum += $between_group_codon_data_hh{$this_analysis}->{$group_name_i}->{$group_name_j}->{$this_product}->{$random_codon_num}->{N_sites};
$sim_S_sites_sum += $between_group_codon_data_hh{$this_analysis}->{$group_name_i}->{$group_name_j}->{$this_product}->{$random_codon_num}->{S_sites};
}
} else { # BOTH CRITERIA 0 OR UNSPECIFIED
$sim_N_diffs_sum += $between_group_codon_data_hh{$this_analysis}->{$group_name_i}->{$group_name_j}->{$this_product}->{$random_codon_num}->{N_diffs};
$sim_S_diffs_sum += $between_group_codon_data_hh{$this_analysis}->{$group_name_i}->{$group_name_j}->{$this_product}->{$random_codon_num}->{S_diffs};
$sim_N_sites_sum += $between_group_codon_data_hh{$this_analysis}->{$group_name_i}->{$group_name_j}->{$this_product}->{$random_codon_num}->{N_sites};
$sim_S_sites_sum += $between_group_codon_data_hh{$this_analysis}->{$group_name_i}->{$group_name_j}->{$this_product}->{$random_codon_num}->{S_sites};
}
} # finished compiling all sampled codons (cols in alignment)
push(@sim_N_diffs_arr,$sim_N_diffs_sum);
push(@sim_S_diffs_arr,$sim_S_diffs_sum);
push(@sim_N_sites_arr,$sim_N_sites_sum);
push(@sim_S_sites_arr,$sim_S_sites_sum);
#print $random_sum . "\n";
} # end last bootstrap
# Recall we have $window_dN_minus_dS above
# CALCULATE BOOTSTRAP STANDARD ERROR HERE; NEI & KUMAR (2000) METHOD
my @sim_dN_minus_dS;
my @sim_dN;
my @sim_dS;
for(my $sim_num = 0; $sim_num < scalar(@sim_N_sites_arr); $sim_num++) {
my $this_round_dN = '*';
if($sim_N_sites_arr[$sim_num] > 0) {
$this_round_dN = $sim_N_diffs_arr[$sim_num] / $sim_N_sites_arr[$sim_num];
}
my $this_round_dS = '*';
if($sim_S_sites_arr[$sim_num] > 0) {
$this_round_dS = $sim_S_diffs_arr[$sim_num] / $sim_S_sites_arr[$sim_num];
}
my $this_round_dN_minus_dS = '*';
if($this_round_dN >= 0 && $this_round_dS >= 0) {
$this_round_dN_minus_dS = $this_round_dN - $this_round_dS;
}
push(@sim_dN_minus_dS, $this_round_dN_minus_dS);
push(@sim_dN, $this_round_dN);
push(@sim_dS, $this_round_dS);
}
$window_SE_dN_minus_dS = &standard_deviation(@sim_dN_minus_dS);
$window_SE_dN = &standard_deviation(@sim_dN);
$window_SE_dS = &standard_deviation(@sim_dS);
if($window_SE_dN_minus_dS > 0) {
$window_boot_Z = $window_dN_minus_dS / $window_SE_dN_minus_dS;
}
if($window_SE_dN_minus_dS == 0) {
$window_SE_dN_minus_dS = 'NA';
}
} # END BOOTSTRAPS for WINDOW
my $product_between_group_dS = '*';
if($between_group_a_p_sums{$this_analysis}->{$this_product}->{S_sites} > 0) {
$product_between_group_dS = $between_group_a_p_sums{$this_analysis}->{$this_product}->{S_diffs} /
$between_group_a_p_sums{$this_analysis}->{$this_product}->{S_sites};
}
my $window_dN_exceeds_product_dS = '';
if($window_dN > $between_product_dS_filtered) {
$window_dN_exceeds_product_dS = '*';
}
# print "\nbetween_product_dS_filtered is $between_product_dS_filtered\n\n";
my $min_defined_codons_in_window = 1000; # or something big
my $window_num_defined_codons_g1 = 0;
for(my $j = $first_codon; $j <= $last_codon; $j++) {
$window_num_defined_codons_g1 += $between_group_codon_data_hh{$this_analysis}->{$group_name_i}->{$group_name_j}->{$this_product}->{$j}->{'num_defined_codons_g1'};
if($between_group_codon_data_hh{$this_analysis}->{$group_name_i}->{$group_name_j}->{$this_product}->{$j}->{'num_defined_codons_g1'} < $min_defined_codons_in_window) {
$min_defined_codons_in_window = $between_group_codon_data_hh{$this_analysis}->{$group_name_i}->{$group_name_j}->{$this_product}->{$j}->{'num_defined_codons_g1'};
}
}
my $window_num_defined_codons_g2 = 0;
for(my $j = $first_codon; $j <= $last_codon; $j++) {
$window_num_defined_codons_g2 += $between_group_codon_data_hh{$this_analysis}->{$group_name_i}->{$group_name_j}->{$this_product}->{$j}->{'num_defined_codons_g2'};
if($between_group_codon_data_hh{$this_analysis}->{$group_name_i}->{$group_name_j}->{$this_product}->{$j}->{'num_defined_codons_g2'} < $min_defined_codons_in_window) {
$min_defined_codons_in_window = $between_group_codon_data_hh{$this_analysis}->{$group_name_i}->{$group_name_j}->{$this_product}->{$j}->{'num_defined_codons_g2'};
}
}
if($min_defined_codons_in_window==0 || $min_defined_codons_in_window eq '' || ! defined($min_defined_codons_in_window)) {
$min_defined_codons_in_window = 0;
}
my $window_mean_num_defined_codons = ($window_num_defined_codons_g1 + $window_num_defined_codons_g2)/2;
# In case filters not passed by ANY codons therein, all results blank
if(! $window_N_sites && ! $window_S_sites && ! $window_N_diffs && ! $window_S_diffs) {
$window_N_sites = 'NA';
$window_S_sites = 'NA';
$window_N_diffs = 'NA';
$window_S_diffs = 'NA';
$window_dN_exceeds_product_dS = 'NA';
}
my $out_line_between_sw = "$this_analysis\t$this_product\t$group_name_i\t$group_name_j\t".
"$window_num\t$first_codon\t$last_codon\t" .
"$window_num_defined_codons_g1\t$window_num_defined_codons_g2\t$window_mean_num_defined_codons\t$min_defined_codons_in_window\t".
"$window_N_sites\t$window_S_sites\t$window_N_diffs\t$window_S_diffs\t".
"$window_dN\t$window_dS\t"; # $window_dN_minus_dS\t$window_w\t$window_dN_exceeds_product_dS";
if($num_bootstraps > 1) {
$out_line_between_sw .= "$window_SE_dN\t$window_SE_dS\t"
}
$out_line_between_sw .= "$window_dN_minus_dS\t$window_w";
if($num_bootstraps > 1) {
my $significance = '';
if($window_SE_dN_minus_dS > 0) {
if(abs($window_boot_Z) > 2.81) {
$significance = '***';
} elsif(abs($window_boot_Z) > 1.96) {
$significance = '**';
} elsif(abs($window_boot_Z) > 1.64) {
$significance = '*';
}
} else {
$significance = 'NA';
}
# Z-tests worthless if only one codon in window
if($sliding_window_size == 1) {
$window_SE_dN_minus_dS = 'NA';
$window_boot_Z = 'NA';
$significance = 'NA';
}
$out_line_between_sw .= "\t$window_SE_dN_minus_dS\t$window_boot_Z\t$significance\n";
} else { # no bootstraps
$out_line_between_sw .= "\n";
}
open(OUTFILE_BETWEEN_SW,">>between\_group\_sw_" . $sliding_window_size . "codons\_results.txt");
print OUTFILE_BETWEEN_SW "$out_line_between_sw";
close OUTFILE_BETWEEN_SW;
} # we're in range of the product
} # finish sliding window step-through
#############################
# BOOTSTRAP THE WHOLE PRODUCT
my $SE_dN_minus_dS = 0;
my $product_boot_Z = 'NA';
my $SE_dN;
my $SE_dS;
my $SE_JC_dN_minus_dS = 0;
my $JC_product_boot_Z = 'NA';
my $SE_JC_dN;
my $SE_JC_dS;
if($num_bootstraps > 1) {
# MAKE BOOTSTRAP FILE
open(BOOT_FILE_PRODUCT,">>$this_product\_bootstrap\_results\.txt");
print BOOT_FILE_PRODUCT "analysis\tgroup1\tgroup2\tproduct_name\tbootstrap_num\tsim_product_N_sites_sum\t".
"sim_product_S_sites_sum\tsim_product_N_diffs_sum\tsim_product_S_diffs_sum\n";
#################
# BOOTSTRAPPING TO CALCULATE STANDARD ERROR HERE?
print "\n#########################################################################################\n" .
"Bootstrapping $this_product between-group $group_name_i vs. $group_name_j for dN/dS standard error...\n";
#print "num_codons_in_product: $num_codons_in_product\n";
my @sim_N_sites_arr;
my @sim_S_sites_arr;
my @sim_N_diffs_arr;
my @sim_S_diffs_arr;
## # PARALLELIZE IT
## mkdir("$product_name\_bootstrap_temp_files");
## chdir("$product_name\_bootstrap_temp_files");
## #my $procs = 60; # number to do at once, cores to assign simultaneously, machine-limited. CUVIER has 80
## my $pm = Parallel::ForkManager->new($procs_per_node);
for(my $bootstrap_num = 1; $bootstrap_num <= $num_bootstraps; $bootstrap_num++) {
## $pm->start and next; # this is IT
srand();
# New bootstrap run, of $num_bootstraps
#print "bootstrap $bootstrap_num\n";
my $sim_product_N_sites_sum = 0;
my $sim_product_S_sites_sum = 0;
my $sim_product_N_diffs_sum = 0;
my $sim_product_S_diffs_sum = 0;
#my $random_sum = 0;
my %sim_poly_site_data_hh; # $sim_poly_site_data_hh{codon_index}->{unique_variants}
# SO, HERE'S the problem: we shouldn't sample WITHIN SITES, but rather SAMPLE
# THE SITES THEMSELVES!!
# (1) indeed, go through $num_codons_in_product
# (2) for each, selection a random codon position's results: Nd, Sd, Ns, Ss.
# where are the aforementioned stored? We've just added the following to do so:
# $codon_to_results_hh{$codon_num}->{N_diffs}
# $codon_to_results_hh{$codon_num}->{S_diffs}
# $codon_to_results_hh{$codon_num}->{N_sites}
# $codon_to_results_hh{$codon_num}->{S_sites}
# SAMPLE codon sites, up to the actual number of codons
for(my $codon_index = 0; $codon_index < $num_sorted_codons; $codon_index++) {
# Which codon site do we choose?
my $random_codon_num = int(rand($num_sorted_codons + 1));
my $num_defined_codons_g1 = $between_group_codon_data_hh{$this_analysis}->{$group_name_i}->{$group_name_j}->{$this_product}->{$random_codon_num}->{'num_defined_codons_g1'};
my $num_defined_codons_g2 = $between_group_codon_data_hh{$this_analysis}->{$group_name_i}->{$group_name_j}->{$this_product}->{$random_codon_num}->{'num_defined_codons_g2'};
if($codon_min_taxa_total > 0 && $codon_min_taxa_group > 0) { # BOTH criteria have been specified
my $taxa_total = $num_defined_codons_g1 + $num_defined_codons_g2;
my $taxa_group_min = $num_defined_codons_g1;
if($num_defined_codons_g2 < $taxa_group_min) {
$taxa_group_min = $num_defined_codons_g2;
}
if($taxa_total >= $codon_min_taxa_total && $taxa_group_min >= $codon_min_taxa_group) { # see if criteria are met by this codon
$sim_product_N_diffs_sum += $between_group_codon_data_hh{$this_analysis}->{$group_name_i}->{$group_name_j}->{$this_product}->{$random_codon_num}->{N_diffs};
$sim_product_S_diffs_sum += $between_group_codon_data_hh{$this_analysis}->{$group_name_i}->{$group_name_j}->{$this_product}->{$random_codon_num}->{S_diffs};
$sim_product_N_sites_sum += $between_group_codon_data_hh{$this_analysis}->{$group_name_i}->{$group_name_j}->{$this_product}->{$random_codon_num}->{N_sites};
$sim_product_S_sites_sum += $between_group_codon_data_hh{$this_analysis}->{$group_name_i}->{$group_name_j}->{$this_product}->{$random_codon_num}->{S_sites};
}
} elsif($codon_min_taxa_total > 0) { # only TOTAL min has been specified
my $taxa_total = $num_defined_codons_g1 + $num_defined_codons_g2;
if($taxa_total >= $codon_min_taxa_total) { # see if criterion is met by this codon
$sim_product_N_diffs_sum += $between_group_codon_data_hh{$this_analysis}->{$group_name_i}->{$group_name_j}->{$this_product}->{$random_codon_num}->{N_diffs};
$sim_product_S_diffs_sum += $between_group_codon_data_hh{$this_analysis}->{$group_name_i}->{$group_name_j}->{$this_product}->{$random_codon_num}->{S_diffs};
$sim_product_N_sites_sum += $between_group_codon_data_hh{$this_analysis}->{$group_name_i}->{$group_name_j}->{$this_product}->{$random_codon_num}->{N_sites};
$sim_product_S_sites_sum += $between_group_codon_data_hh{$this_analysis}->{$group_name_i}->{$group_name_j}->{$this_product}->{$random_codon_num}->{S_sites};
}
} elsif($codon_min_taxa_group > 0) { # only GROUP min has been specified
my $taxa_group_min = $num_defined_codons_g1;
if($num_defined_codons_g2 < $taxa_group_min) {
$taxa_group_min = $num_defined_codons_g2;
}
if($taxa_group_min >= $codon_min_taxa_group) { # see if criterion is met by this codon
$sim_product_N_diffs_sum += $between_group_codon_data_hh{$this_analysis}->{$group_name_i}->{$group_name_j}->{$this_product}->{$random_codon_num}->{N_diffs};
$sim_product_S_diffs_sum += $between_group_codon_data_hh{$this_analysis}->{$group_name_i}->{$group_name_j}->{$this_product}->{$random_codon_num}->{S_diffs};
$sim_product_N_sites_sum += $between_group_codon_data_hh{$this_analysis}->{$group_name_i}->{$group_name_j}->{$this_product}->{$random_codon_num}->{N_sites};
$sim_product_S_sites_sum += $between_group_codon_data_hh{$this_analysis}->{$group_name_i}->{$group_name_j}->{$this_product}->{$random_codon_num}->{S_sites};
}
} else { # BOTH CRITERIA 0 OR UNSPECIFIED
$sim_product_N_diffs_sum += $between_group_codon_data_hh{$this_analysis}->{$group_name_i}->{$group_name_j}->{$this_product}->{$random_codon_num}->{N_diffs};
$sim_product_S_diffs_sum += $between_group_codon_data_hh{$this_analysis}->{$group_name_i}->{$group_name_j}->{$this_product}->{$random_codon_num}->{S_diffs};
$sim_product_N_sites_sum += $between_group_codon_data_hh{$this_analysis}->{$group_name_i}->{$group_name_j}->{$this_product}->{$random_codon_num}->{N_sites};
$sim_product_S_sites_sum += $between_group_codon_data_hh{$this_analysis}->{$group_name_i}->{$group_name_j}->{$this_product}->{$random_codon_num}->{S_sites};
}
} # finished compiling all sampled codons (cols in alignment)
# Print product totals for BOOTSTRAP
my $out_line_boot = "ANALYSIS\t$group_name_i\t$group_name_j\t$this_product\t$bootstrap_num\t$sim_product_N_sites_sum\t".
"$sim_product_S_sites_sum\t$sim_product_N_diffs_sum\t$sim_product_S_diffs_sum\n";
print BOOT_FILE_PRODUCT "$out_line_boot";
#print "$out_line_boot";
push(@sim_N_diffs_arr,$sim_product_N_diffs_sum);
push(@sim_S_diffs_arr,$sim_product_S_diffs_sum);
push(@sim_N_sites_arr,$sim_product_N_sites_sum);
push(@sim_S_sites_arr,$sim_product_S_sites_sum);
#print $random_sum . "\n";
} # end last bootstrap
close BOOT_FILE_PRODUCT;
# Calculate ACTUAL overall values for the product
# my $between_product_dN_filtered;
# if($between_product_N_sites_sum_filtered > 0) {
# $between_product_dN_filtered = $between_product_N_diffs_sum_filtered / $between_product_N_sites_sum_filtered;
# } else {
# $between_product_dN_filtered = '*';
# }
#