/
OLGenie.pl
executable file
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OLGenie.pl
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#! /usr/bin/env perl
# PROGRAM: SNPGenie + Wei-Zhang method for overlapping genes (overlapgenie; OLGenie)
# using codon-based analysis for dNN/dSN/dNS/dSS
#########################################################################################
# EXAMPLE CALL:
#########################################################################################
# OLGenie.pl --fasta_file=all_seqs.fasta --frame=sas11 --verbose > OLGenie_log.txt
#########################################################################################
#########################################################################################
## 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/>.
#########################################################################################
# AUTHOR: Chase W. Nelson
# Copyright (C) 2019 Chase W. Nelson
# DATE CREATED: April 2019
# CONTACT1: cnelson@amnh.org
# CONTACT2: cwnelson88@gmail.com
# AFFILIATION: Sackler Institute for Comparative Genomics, American Museum of Natural
# History, New York, NY 10024, USA
# CITATION1: OLGenie, https://github.com/chasewnelson/OLGenie
# CITATION2: Nelson CW, Ardern Z, Wei X. OLGenie: detecting natural selection to identify functional overlapping genes. In preparation.
# 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 Data::Dumper;
use List::Util qw(max);
use Getopt::Long;
# Get the time
my $time1 = time;
my $local_time1 = localtime;
STDOUT->autoflush(1);
my @nucleotides = qw/A C G T/;
#########################################################################################
# INITIALIZE (OPTIONAL) INPUT VARIABLES
my $fasta_file;
my $frame;
my $output_file;
my $mode;
my $verbose;
my $easter;
my $verbose_messages; # only meant for developing code
my $die_message = "\n\n" .
"################################################################################\n" .
"### OLGenie for analysis of selection in overlapping genes using member pairs.\n" .
"################################################################################\n\n" .
"\n################################################################################\n" .
"### OPTIONS:\n" .
"################################################################################\n" .
"\n" .
"\t--fasta_file (REQUIRED): a FASTA file containing multiple aligned sequences of one coding sequence.\n" .
"\t\tThe entire coding sequence must be an overlapping gene (OLG), with no non-overlapping codons.\n" .
"\t\tThe frame must be the frame of the reference gene (ORF1). (See the --frame option.)\n\n" .
"\t--frame (REQUIRED): the frame of the overlapping gene (OLG) relationship: ss12, ss13, sas11, sas12, or sas13:\n\n" .
"\t\tSENSE-SENSE:\n" .
"\t\t\tss12:\n\t\t\tORF1: 1-2-3-1-2-3-1\n\t\t\tORF2: 2-3-1-2-3-1-2\n" .
"\t\t\tss13:\n\t\t\tORF1: 1-2-3-1-2-3-1\n\t\t\tORF2: 3-1-2-3-1-2-3\n\n ".
"\t\tSENSE-ANTISENSE:\n" .
"\t\t\tsas11:\n\t\t\tORF1: 1-2-3-1-2-3-1\n\t\t\tORF2: 1-3-2-1-3-2-1\n" .
"\t\t\tsas12:\n\t\t\tORF1: 1-2-3-1-2-3-1\n\t\t\tORF2: 2-1-3-2-1-3-2\n" .
"\t\t\tsas13:\n\t\t\tORF1: 1-2-3-1-2-3-1\n\t\t\tORF2: 3-2-1-3-2-1-3\n\n\n" .
"\t--output_file (OPTIONAL): name of the TAB-delimited output file to be placed in the working directory\n" .
"\t\tunless a full path name is given. If not specified, a file will be printed in the working directory\n" .
"\t\tby the name OLGenie_codon_results.txt (DEFAULT).\n\n" .
"\t--verbose (OPTIONAL): tell OLGenie to report all unique nonamers (9-mers) overlapping each reference\n" .
"\t\tcodon, along with their counts, in the output file. May lead to large output files in cases with\n" .
"\t\tmany and/or divergent sequences.\n\n" .
"\n################################################################################\n" .
"### EXAMPLE:\n" .
"################################################################################\n\n" .
"\t\$ OLGenie.pl --fasta_file=my_alignment.fasta --frame=ss13 --output_file=OLGenie_codon_results.txt --verbose\n\n" .
"################################################################################\n\n";
# Get user input, if given. If a Boolean argument is passed, its value is 1; else undef
GetOptions( "fasta_file=s" => \$fasta_file,
"frame=s" => \$frame,
"output_file=s" => \$output_file,
"mode=s" => \$mode,
"verbose" => \$verbose,
"easter" => \$easter,
"verbose_messages" => \$verbose_messages )
or die $die_message;
# If an argument is called as a flag, its value is 0; if not called, it's null
unless(-f "$fasta_file" &&
($frame eq 'ss12' || $frame eq 'ss13' || $frame eq 'sas11' || $frame eq 'sas12' || $frame eq 'sas13')) {
die $die_message;
}
my $fasta_file_short = $fasta_file;
$fasta_file_short =~ s/(.*\/)?(.+\.\w+)/$2/;
unless("$output_file") {
$output_file = "OLGenie\_codon\_results\.txt";
}
if($mode) {
if($mode ne 'within_group' && $mode ne 'between_group') {
die $die_message;
}
}
unless("$mode") {
$mode = 'within_group';
}
print "\n################################################################################" .
"\n## ##" .
"\n## OLGenie Initiated! ##" .
"\n## ##" .
"\n################################################################################\n";
print "\nOLGenie initiated at local time $local_time1\n";
print "\n################################################################################";
print "\nANALYSIS MODE=$mode\...\n";
# Read in the group of sequences from the fasta file
my %header2sequence;
my $seq = '';
my $header = '';
my @headers_arr;
my $seq_num = 0;
my $last_seq_length;
open(IN_FASTA, "$fasta_file") or die "Could not open file $fasta_file\n";
print "\n################################################################################";
print "\nRecording coding sequence data for $fasta_file...\n";
while(<IN_FASTA>) {
chomp;
if(/>/) {
if($seq_num == 0) {
$header = $_;
$header =~ s/^>//; # get rid of FASTA header indicator
$header =~ tr/\|/_/; # convert pipes (|) to underscores (_), following IQTree convention
$header =~ s/\s.*$//; # trim anything including and after the first whitespace
$seq_num ++;
} else {
$seq = uc($seq);
$seq =~ tr/U/T/;
if($header =~ /[^\w^\-^\.]/) {
die "\n### TAXA NAMES CONTAIN INAPPROPRIATE CHARACTERS IN FASTA FILE: $header.\n" .
"### Only alphanumeric characters (a-z, A-Z, 0-9), underscores (_), dashes (-), and periods (.) may be used. SCRIPT TERMINATED.\n\n";
}
if(exists $header2sequence{$header}) {
die "\n\n### DIE: Each sequence in the FASTA multiple sequence alignment must have a unique header (up to first whitespace) for identification. TERMINATED.\n\n";
} else {
$header2sequence{$header} = $seq;
push(@headers_arr, $header);
}
$header = $_;
$header =~ s/^>//; # get rid of FASTA header indicator
$header =~ tr/\|/_/; # convert pipes (|) to underscores (_), following IQTree convention
$header =~ s/\s.*$//; # trim anything including and after the first whitespace
$seq_num ++;
my $this_seq_length = length($seq);
unless($this_seq_length % 3 == 0) {
die "\n\n### DIE: Sequences must be a complete set of codons, i.e., the nucleotide length".
"### must be evenly divisible by 3. Instead, the length is $this_seq_length\. TERMINATED.\n\n";
}
if($last_seq_length && ($last_seq_length != $this_seq_length)) {
die "\n\n### DIE: The sequences must be aligned, i.e., must be the same length. TERMINATED.\n\n";
} else {
$last_seq_length = $this_seq_length;
$seq = '';
}
}
} else {
$seq .= $_;
}
}
close IN_FASTA;
$seq = uc($seq);
$seq =~ tr/U/T/;
if($header =~ /^[^\w^\-^\.]/) {
die "\n### TAXA NAMES CONTAIN INAPPROPRIATE CHARACTERS IN FASTA FILE: $header.\n" .
"### Only alphanumeric characters (a-z, A-Z, 0-9), underscores (_), dashes (-), and periods (.) may be used. SCRIPT TERMINATED.\n\n";
}
$header2sequence{$header} = $seq;
push(@headers_arr, $header);
##########################################################################################
# Store the sequence index of each FASTA header
#my %header_to_index;
my %header_to_def_len;
foreach my $curr_header (keys %header2sequence) {
# Get seq
my $seq = $header2sequence{$curr_header};
# Count undefined length
my $num_N = $seq =~ s/N//g;
my $num_gap = $seq =~ s/-//g;
my $defined_length = $last_seq_length - $num_N - $num_gap;
$header_to_def_len{$curr_header} = $defined_length;
if($verbose_messages) { print "seq $header has a defined length of $defined_length\n" }
}
##########################################################################################
# Print which frame
print "\n################################################################################";
print "\nAnalyzing overlapping gene in frame $frame\...\n";
##########################################################################################
# DETERMINE ALL UNIQUE 9-mers along the alignment
my $print_codon_header = 0;
# IF PAIRWISE MODE ACTIVATED, record results separately for each pair
if($mode eq 'between_group') {
print "\n################################################################################";
print "\nActivating between-group mode (pairwise comparisons between user-defined groups)\...\n";
for(my $i = 0; $i < @headers_arr; $i++) {
my $curr_header_1 = $headers_arr[$i];
my $curr_seq_1 = $header2sequence{$curr_header_1};
for(my $k = $i + 1; $k < @headers_arr; $k++) {
my $curr_header_2 = $headers_arr[$k];
my $curr_seq_2 = $header2sequence{$curr_header_2};
# We've already made sure the sequence is a multiple of 3; loop codons
my %unique_9mers;
for(my $j = 0; $j <= length($curr_seq_1) - 9; $j += 3) {
$unique_9mers{$j}->{substr($curr_seq_1, $j, 9)}++; # increment current 9-mer count
$unique_9mers{$j}->{substr($curr_seq_2, $j, 9)}++; # increment current 9-mer count
}
# The current pair's data have been placed in %unique_9mers; call method
&olgenie_method(\%unique_9mers, $curr_header_1, $curr_header_2);
}
}
} elsif($mode eq 'within_group') { # don't calculate all pairs separately
print "\n################################################################################";
print "\nActivating within-group mode (all pairwise comparisons)\...\n";
my %unique_9mers;
for(my $i = 0; $i < @headers_arr; $i++) {
my $curr_header = $headers_arr[$i];
my $curr_seq = $header2sequence{$curr_header};
# We've already made sure the sequence is a multiple of 3
for(my $j = 0; $j <= length($curr_seq) - 9; $j += 3) {
$unique_9mers{$j}->{substr($curr_seq, $j, 9)}++; # increment current 9-mer count
}
}
# The whole alignments data have been placed in %unique_9mers; call method
&olgenie_method(\%unique_9mers);
}
sub olgenie_method {
my($unique_9mers_ref, $seq_1, $seq_2) = @_;
# if($verbose) { print "We got the arguments: @_\n" }
my %unique_9mers;
if($unique_9mers_ref ne '') {
%unique_9mers = %{$unique_9mers_ref};
}
my $seqs_compared;
if(! $seq_1 && ! $seq_2) {
$seqs_compared = 'all';
} else {
$seqs_compared = "$seq_1\:$seq_2";
}
if($verbose_messages) {
foreach my $site (sort {$a <=> $b} keys %unique_9mers) {
print "SITE: " . ($site + 1) . "-" . ($site + 9) . "\n";
foreach my $nonamer (sort keys %{$unique_9mers{$site}}) {
print "$nonamer\: " . $unique_9mers{$site}->{$nonamer} . "\n";
}
print "\n";
}
}
##########################################################################################
# ANALYZE ALL UNIQUE PAIRS, PRINT CODON FILE, AND STORE TOTALS
my %site_diffs_hh;
my %seq2sites;
my %seq_completed;
print "\n################################################################################".
"\nANALYZING SEQUENCE PAIR(S): $seqs_compared\n";
my %codon_data_hh;
# LOOP ALL NONAMER POSITIONS
foreach my $seq_site_index (sort {$a <=> $b} keys %unique_9mers) {
if($verbose_messages) { print "\n\n###seq_site_index=$seq_site_index\n" }
### NEW PHYLOGENY-NAIVE APPROACH
my @nonamers_sorted = sort keys %{$unique_9mers{$seq_site_index}};
my $codon_in_seq = ($seq_site_index + 6) / 3; # start with codon 2 (middle of first nonamer)
my $comparisons_sum = 0;
my $NN_sites_numerator = 0;
my $SN_sites_numerator = 0;
my $NS_sites_numerator = 0;
my $SS_sites_numerator = 0;
my $NN_diffs_numerator = 0;
my $SN_diffs_numerator = 0;
my $NS_diffs_numerator = 0;
my $SS_diffs_numerator = 0;
my $this_codon_MNV = 'FALSE';
my %ref_codons_to_counts;
my %alt1_codons_to_counts;
my %alt2_codons_to_counts;
for(my $nonamer1_index = 0; $nonamer1_index < @nonamers_sorted; $nonamer1_index++) {
my $nonamer1 = $nonamers_sorted[$nonamer1_index];
my $nonamer1_count = $unique_9mers{$seq_site_index}->{$nonamer1};
$codon_data_hh{$codon_in_seq}->{nonamers} .= "$nonamer1\:";
$codon_data_hh{$codon_in_seq}->{nonamer_counts} .= "$nonamer1_count\:";
$ref_codons_to_counts{substr($nonamer1, 3, 3)} += $nonamer1_count; # middle codon
for(my $nonamer2_index = $nonamer1_index; $nonamer2_index < @nonamers_sorted; $nonamer2_index++) { # start with SELF and proceed
my $nonamer2 = $nonamers_sorted[$nonamer2_index];
if($verbose_messages) { print "\n#nonamer pair: $nonamer1_index vs. $nonamer2_index\n" }
# EXAMINE THE PROVIDED PRODUCT FOR THE CURRENT MEMBER PAIR
my $nonamer1_len = length($nonamer1);
my $nonamer2_len = length($nonamer2);
if(($nonamer1_len % 3) != 0) {
die "\n\nDIE: sequence of $nonamer1 is not a multiple of 3 (complete codon set). TERMINATED.\n\n";
}
if(($nonamer2_len % 3) != 0) {
die "\n\nDIE: sequence of in $nonamer2 is not a multiple of 3 (complete codon set). TERMINATED.\n\n";
}
if($nonamer1_len != $nonamer2_len) {
die "\n\nDIE: The length of sequence is different in $nonamer1 and $nonamer2\. TERMINATED.\n\n";
}
my $num_codons = $nonamer1_len / 3;
if($verbose_messages) { print "num_codons=$num_codons\n" }
# STORE CODONS for nonamer1 and nonamer2 here; we've verified they're the same length
#NONAMER_CODONS: for(my $codon_num = 1; $codon_num <= $num_codons; $codon_num++) { # each ORF1 codon
my $codon_num = 2; # with the new approach, it's always the central/focal/ref codon
my $codon_index = $codon_num - 1;
my $site_index = 0 + (3 * $codon_index);
# Extract codons for ORF1
my $codon_nonamer1_ORF1 = substr($nonamer1, $site_index, 3);
my $codon_nonamer2_ORF1 = substr($nonamer2, $site_index, 3);
my $AA_nonamer1_ORF1 = get_amino_acid($codon_nonamer1_ORF1);
my $AA_nonamer2_ORF1 = get_amino_acid($codon_nonamer2_ORF1);
if($verbose_messages) {
print "codon_nonamer1_ORF1=$codon_nonamer1_ORF1\n";
print "codon_nonamer2_ORF1=$codon_nonamer2_ORF1\n";
print "AA_nonamer1_ORF1=$AA_nonamer1_ORF1\n";
print "AA_nonamer2_ORF1=$AA_nonamer2_ORF1\n";
}
if($codon_num < $num_codons && ($AA_nonamer1_ORF1 eq '*' || $AA_nonamer2_ORF1 eq '*')) {
print "### WARNING! ORF1, $nonamer1\-$nonamer2 comparison, codon $codon_in_seq encodes a within-frame STOP codon. Wrong frame selection ($frame)?\n";
}
# IF UNDEFINED CODONS, NOTHING AT ALL CAN BE DETERMINED
unless($codon_nonamer1_ORF1 =~ /N/ || $codon_nonamer2_ORF1 =~ /N/ || $codon_nonamer1_ORF1 =~ /-/ ||$codon_nonamer2_ORF1 =~ /-/) {
# Extract codons for (OVERLAPPING) ORF2
##################################################################################################
##################### SENSE-SENSE:
##################### ss12:
##################### ORF1: 1-2-3-1-2-3-1
##################### ORF2: 2-3-1-2-3-1-2
##################################################################################################
if($frame eq 'ss12') {
# Last 2 nt of prev codon, first 1 nt of next codon (same strand)
my $codon_nonamer1_ORF2_prev = substr($nonamer1, ($site_index - 1), 3);
my $codon_nonamer1_ORF2_next = substr($nonamer1, ($site_index + 2), 3);
my $codon_nonamer2_ORF2_prev = substr($nonamer2, ($site_index - 1), 3);
my $codon_nonamer2_ORF2_next = substr($nonamer2, ($site_index + 2), 3);
# COMEBACK: speed could be improved by re-using last round's 'codon' and '*_next'
if($verbose_messages) {
print "site_index=$site_index\n";
print "nonamer1=$nonamer1\n";
print "nonamer2=$nonamer2\n";
print "nonamer1_count=$nonamer1_count\n";
print "codon_nonamer1_ORF2_prev=$codon_nonamer1_ORF2_prev\n";
print "codon_nonamer1_ORF2_next=$codon_nonamer1_ORF2_next\n";
print "codon_nonamer2_ORF2_prev=$codon_nonamer2_ORF2_prev\n";
print "codon_nonamer2_ORF2_next=$codon_nonamer2_ORF2_next\n";
}
my $AA_nonamer1_ORF2_prev = get_amino_acid($codon_nonamer1_ORF2_prev);
my $AA_nonamer1_ORF2_next = get_amino_acid($codon_nonamer1_ORF2_next);
my $AA_nonamer2_ORF2_prev = get_amino_acid($codon_nonamer2_ORF2_prev);
my $AA_nonamer2_ORF2_next = get_amino_acid($codon_nonamer2_ORF2_next);
if($codon_num > 2 && ($AA_nonamer1_ORF2_prev eq '*' || $AA_nonamer1_ORF2_next eq '*' || $AA_nonamer2_ORF2_prev eq '*' || $AA_nonamer2_ORF2_next eq '*')) {
print "### WARNING! ORF2, $nonamer1\-$nonamer2 comparison, near ORF1 codon $codon_in_seq encodes a within-frame STOP codon. Wrong frame selection ($frame)?\n";
}
#######################
# GET NUMBER OF SITES
if($codon_num > 1 && $codon_num < $num_codons) {
# both ORF2's PREV and NEXT codons fully defined, 3 sites to examine
# New phylogeny-naive approach
$alt1_codons_to_counts{$codon_nonamer1_ORF2_prev} += $nonamer1_count;
$alt1_codons_to_counts{$codon_nonamer2_ORF2_prev} += $nonamer1_count; # ADDED
$alt2_codons_to_counts{$codon_nonamer1_ORF2_next} += $nonamer1_count;
$alt2_codons_to_counts{$codon_nonamer2_ORF2_next} += $nonamer1_count; # ADDED
my $nonamer1_num_changes_poss_site1 = 0;
my $nonamer1_num_changes_NN_site1 = 0;
my $nonamer1_num_changes_SN_site1 = 0;
my $nonamer1_num_changes_NS_site1 = 0;
my $nonamer1_num_changes_SS_site1 = 0;
my $nonamer1_num_changes_poss_site2 = 0;
my $nonamer1_num_changes_NN_site2 = 0;
my $nonamer1_num_changes_SN_site2 = 0;
my $nonamer1_num_changes_NS_site2 = 0;
my $nonamer1_num_changes_SS_site2 = 0;
my $nonamer1_num_changes_poss_site3 = 0;
my $nonamer1_num_changes_NN_site3 = 0;
my $nonamer1_num_changes_SN_site3 = 0;
my $nonamer1_num_changes_NS_site3 = 0;
my $nonamer1_num_changes_SS_site3 = 0;
my $nonamer2_num_changes_poss_site1 = 0;
my $nonamer2_num_changes_NN_site1 = 0;
my $nonamer2_num_changes_SN_site1 = 0;
my $nonamer2_num_changes_NS_site1 = 0;
my $nonamer2_num_changes_SS_site1 = 0;
my $nonamer2_num_changes_poss_site2 = 0;
my $nonamer2_num_changes_NN_site2 = 0;
my $nonamer2_num_changes_SN_site2 = 0;
my $nonamer2_num_changes_NS_site2 = 0;
my $nonamer2_num_changes_SS_site2 = 0;
my $nonamer2_num_changes_poss_site3 = 0;
my $nonamer2_num_changes_NN_site3 = 0;
my $nonamer2_num_changes_SN_site3 = 0;
my $nonamer2_num_changes_NS_site3 = 0;
my $nonamer2_num_changes_SS_site3 = 0;
my $NN_diffs = 0;
my $SN_diffs = 0;
my $NS_diffs = 0;
my $SS_diffs = 0;
# Just ORF1 (reference)
my $nt1_nonamer1_WT = substr($codon_nonamer1_ORF1, 0, 1);
my $nt2_nonamer1_WT = substr($codon_nonamer1_ORF1, 1, 1);
my $nt3_nonamer1_WT = substr($codon_nonamer1_ORF1, 2, 1);
my $nt1_nonamer2_WT = substr($codon_nonamer2_ORF1, 0, 1);
my $nt2_nonamer2_WT = substr($codon_nonamer2_ORF1, 1, 1);
my $nt3_nonamer2_WT = substr($codon_nonamer2_ORF1, 2, 1);
foreach my $nt (@nucleotides) {
##################################################################
# nonamer1
# SITE 1
# What is each change to ORF1 CODON SITE 1 / ORF2 CODON SITE 2?
if($nt ne $nt1_nonamer1_WT) { # only one possibility for this site
# only one possibility for this site
my $nonamer1_STOP_caused = 0;
my $codon_nonamer1_ORF1_MUT = $codon_nonamer1_ORF1;
$codon_nonamer1_ORF1_MUT =~ s/[ACGT]([ACGT])([ACGT])/$nt$1$2/;
my $codon_nonamer1_ORF2_prev_MUT = $codon_nonamer1_ORF2_prev;
$codon_nonamer1_ORF2_prev_MUT =~ s/([ACGT])[ACGT]([ACGT])/$1$nt$2/;
my $nonamer1_ORF1_effect = 'S';
#nonamer1-ORF1
my $AA_nonamer1_ORF1_MUT = get_amino_acid($codon_nonamer1_ORF1_MUT);
if($AA_nonamer1_ORF1_MUT ne $AA_nonamer1_ORF1) {
$nonamer1_ORF1_effect = 'N';
}
if($AA_nonamer1_ORF1 eq '*' || $AA_nonamer1_ORF1_MUT eq '*') {
$nonamer1_STOP_caused++;
}
my $nonamer1_ORF2_prev_effect = 'S';
#nonamer1-ORF2
my $AA_nonamer1_ORF2_prev_MUT = get_amino_acid($codon_nonamer1_ORF2_prev_MUT);
if($AA_nonamer1_ORF2_prev_MUT ne $AA_nonamer1_ORF2_prev) {
$nonamer1_ORF2_prev_effect = 'N';
}
if($AA_nonamer1_ORF2_prev eq '*' || $AA_nonamer1_ORF2_prev_MUT eq '*') {
$nonamer1_STOP_caused++;
}
# EASTER: enumerate all possible, site 1
if($easter) { print "$codon_in_seq\tnonamer1\t$codon_nonamer1_ORF1\t$codon_nonamer1_ORF2_prev\t$nt1_nonamer1_WT\t$nt\t$AA_nonamer1_ORF1\t$AA_nonamer1_ORF2_prev\t1\t$AA_nonamer1_ORF1_MUT\t$AA_nonamer1_ORF2_prev_MUT\n" }
# TALLY VIABLE CHANGES, GET NUMBER OF DIFFS
unless($nonamer1_STOP_caused > 0) {
$nonamer1_num_changes_poss_site1++;
if($nonamer1_ORF1_effect eq 'N') {
if($nonamer1_ORF2_prev_effect eq 'N') {
$nonamer1_num_changes_NN_site1++;
# ACTUAL DIFF
if($nt eq $nt1_nonamer2_WT) {
$NN_diffs++;
$site_diffs_hh{$codon_num}->{1}->{NN_diffs}++;
}
} elsif($nonamer1_ORF2_prev_effect eq 'S') {
$nonamer1_num_changes_NS_site1++;
# ACTUAL DIFF
if($nt eq $nt1_nonamer2_WT) {
$NS_diffs++;
$site_diffs_hh{$codon_num}->{1}->{NS_diffs}++;
}
}
} elsif($nonamer1_ORF1_effect eq 'S') {
if($nonamer1_ORF2_prev_effect eq 'N') {
$nonamer1_num_changes_SN_site1++;
# ACTUAL DIFF
if($nt eq $nt1_nonamer2_WT) {
$SN_diffs++;
$site_diffs_hh{$codon_num}->{1}->{SN_diffs}++;
}
} elsif($nonamer1_ORF2_prev_effect eq 'S') {
$nonamer1_num_changes_SS_site1++;
# ACTUAL DIFF
if($nt eq $nt1_nonamer2_WT) {
$SS_diffs++;
$site_diffs_hh{$codon_num}->{1}->{SS_diffs}++;
}
}
}
}
} # end member 1 site 1
# SITE 2
# What is each change to ORF1 CODON SITE 2 / ORF2 CODON SITE 3?
if($nt ne $nt2_nonamer1_WT) { # only one possibility for this site
my $nonamer1_STOP_caused = 0;
my $codon_nonamer1_ORF1_MUT = $codon_nonamer1_ORF1;
$codon_nonamer1_ORF1_MUT =~ s/([ACGT])[ACGT]([ACGT])/$1$nt$2/;
my $codon_nonamer1_ORF2_prev_MUT = $codon_nonamer1_ORF2_prev;
$codon_nonamer1_ORF2_prev_MUT =~ s/([ACGT])([ACGT])[ACGT]/$1$2$nt/;
my $nonamer1_ORF1_effect = 'S';
#nonamer1-ORF1
my $AA_nonamer1_ORF1_MUT = get_amino_acid($codon_nonamer1_ORF1_MUT);
if($AA_nonamer1_ORF1_MUT ne $AA_nonamer1_ORF1) {
$nonamer1_ORF1_effect = 'N';
}
if($AA_nonamer1_ORF1 eq '*' || $AA_nonamer1_ORF1_MUT eq '*') {
$nonamer1_STOP_caused++;
}
my $nonamer1_ORF2_prev_effect = 'S';
#nonamer1-ORF2
my $AA_nonamer1_ORF2_prev_MUT = get_amino_acid($codon_nonamer1_ORF2_prev_MUT);
if($AA_nonamer1_ORF2_prev_MUT ne $AA_nonamer1_ORF2_prev) {
$nonamer1_ORF2_prev_effect = 'N';
}
if($AA_nonamer1_ORF2_prev eq '*' || $AA_nonamer1_ORF2_prev_MUT eq '*') {
$nonamer1_STOP_caused++;
}
# EASTER: enumerate all possible, site 2
if($easter) { print "$codon_in_seq\tnonamer1\t$codon_nonamer1_ORF1\t$codon_nonamer1_ORF2_prev\t$nt2_nonamer1_WT\t$nt\t$AA_nonamer1_ORF1\t$AA_nonamer1_ORF2_prev\t2\t$AA_nonamer1_ORF1_MUT\t$AA_nonamer1_ORF2_prev_MUT\n" }
# TALLY VIABLE CHANGES, GET NUMBER OF DIFFS
unless($nonamer1_STOP_caused > 0) {
$nonamer1_num_changes_poss_site2++;
if($nonamer1_ORF1_effect eq 'N') {
if($nonamer1_ORF2_prev_effect eq 'N') {
$nonamer1_num_changes_NN_site2++;
# ACTUAL DIFF
if($nt eq $nt2_nonamer2_WT) {
$NN_diffs++;
$site_diffs_hh{$codon_num}->{2}->{NN_diffs}++;
}
} elsif($nonamer1_ORF2_prev_effect eq 'S') {
$nonamer1_num_changes_NS_site2++;
# ACTUAL DIFF
if($nt eq $nt2_nonamer2_WT) {
$NS_diffs++;
$site_diffs_hh{$codon_num}->{2}->{NS_diffs}++;
}
}
} elsif($nonamer1_ORF1_effect eq 'S') {
if($nonamer1_ORF2_prev_effect eq 'N') {
$nonamer1_num_changes_SN_site2++;
# ACTUAL DIFF
if($nt eq $nt2_nonamer2_WT) {
$SN_diffs++;
$site_diffs_hh{$codon_num}->{2}->{SN_diffs}++;
}
} elsif($nonamer1_ORF2_prev_effect eq 'S') {
$nonamer1_num_changes_SS_site2++;
# ACTUAL DIFF
if($nt eq $nt2_nonamer2_WT) {
$SS_diffs++;
$site_diffs_hh{$codon_num}->{2}->{SS_diffs}++;
}
}
}
}
} # end member 1 site 2
# SITE 3
# What is each change to ORF1 CODON SITE 3 / ORF2 (next) CODON SITE 1?
if($nt ne $nt3_nonamer1_WT) { # only one possibility for this site
my $nonamer1_STOP_caused = 0;
my $codon_nonamer1_ORF1_MUT = $codon_nonamer1_ORF1;
$codon_nonamer1_ORF1_MUT =~ s/([ACGT])([ACGT])[ACGT]/$1$2$nt/;
my $codon_nonamer1_ORF2_next_MUT = $codon_nonamer1_ORF2_next;
$codon_nonamer1_ORF2_next_MUT =~ s/[ACGT]([ACGT])([ACGT])/$nt$1$2/;
my $nonamer1_ORF1_effect = 'S';
#nonamer1-ORF1
my $AA_nonamer1_ORF1_MUT = get_amino_acid($codon_nonamer1_ORF1_MUT);
if($AA_nonamer1_ORF1_MUT ne $AA_nonamer1_ORF1) {
$nonamer1_ORF1_effect = 'N';
}
if($AA_nonamer1_ORF1 eq '*' || $AA_nonamer1_ORF1_MUT eq '*') {
$nonamer1_STOP_caused++;
}
my $nonamer1_ORF2_next_effect = 'S';
#nonamer1-ORF2
my $AA_nonamer1_ORF2_next_MUT = get_amino_acid($codon_nonamer1_ORF2_next_MUT);
if($AA_nonamer1_ORF2_next_MUT ne $AA_nonamer1_ORF2_next) {
$nonamer1_ORF2_next_effect = 'N';
}
if($AA_nonamer1_ORF2_next eq '*' || $AA_nonamer1_ORF2_next_MUT eq '*') {
$nonamer1_STOP_caused++;
}
# EASTER: enumerate all possible, site 3
if($easter) { print "$codon_in_seq\tnonamer1\t$codon_nonamer1_ORF1\t$codon_nonamer1_ORF2_next\t$nt3_nonamer1_WT\t$nt\t$AA_nonamer1_ORF1\t$AA_nonamer1_ORF2_next\t3\t$AA_nonamer1_ORF1_MUT\t$AA_nonamer1_ORF2_next_MUT\n" }
# TALLY VIABLE CHANGES, GET NUMBER OF DIFFS
unless($nonamer1_STOP_caused > 0) {
$nonamer1_num_changes_poss_site3++;
if($nonamer1_ORF1_effect eq 'N') {
if($nonamer1_ORF2_next_effect eq 'N') {
$nonamer1_num_changes_NN_site3++;
# ACTUAL DIFF
if($nt eq $nt3_nonamer2_WT) {
$NN_diffs++;
$site_diffs_hh{$codon_num}->{3}->{NN_diffs}++;
}
} elsif($nonamer1_ORF2_next_effect eq 'S') {
$nonamer1_num_changes_NS_site3++;
# ACTUAL DIFF
if($nt eq $nt3_nonamer2_WT) {
$NS_diffs++;
$site_diffs_hh{$codon_num}->{3}->{NS_diffs}++;
}
}
} elsif($nonamer1_ORF1_effect eq 'S') {
if($nonamer1_ORF2_next_effect eq 'N') {
$nonamer1_num_changes_SN_site3++;
# ACTUAL DIFF
if($nt eq $nt3_nonamer2_WT) {
$SN_diffs++;
$site_diffs_hh{$codon_num}->{3}->{SN_diffs}++;
}
} elsif($nonamer1_ORF2_next_effect eq 'S') {
$nonamer1_num_changes_SS_site3++;
# ACTUAL DIFF
if($nt eq $nt3_nonamer2_WT) {
$SS_diffs++;
$site_diffs_hh{$codon_num}->{3}->{SS_diffs}++;
}
}
}
}
} # end member 1 site 3
##################################################################
# nonamer2
# SITE 1
# What is each change to ORF1 CODON SITE 1 / ORF2 CODON SITE 2?
if($nt ne $nt1_nonamer2_WT) { # only one possibility for this site
# only one possibility for this site
my $nonamer2_STOP_caused = 0;
my $codon_nonamer2_ORF1_MUT = $codon_nonamer2_ORF1;
$codon_nonamer2_ORF1_MUT =~ s/[ACGT]([ACGT])([ACGT])/$nt$1$2/;
my $codon_nonamer2_ORF2_prev_MUT = $codon_nonamer2_ORF2_prev;
$codon_nonamer2_ORF2_prev_MUT =~ s/([ACGT])[ACGT]([ACGT])/$1$nt$2/;
my $nonamer2_ORF1_effect = 'S';
#nonamer1-ORF1
my $AA_nonamer2_ORF1_MUT = get_amino_acid($codon_nonamer2_ORF1_MUT);
if($AA_nonamer2_ORF1_MUT ne $AA_nonamer2_ORF1) {
$nonamer2_ORF1_effect = 'N';
}
if($AA_nonamer2_ORF1 eq '*' || $AA_nonamer2_ORF1_MUT eq '*') {
$nonamer2_STOP_caused++;
}
my $nonamer2_ORF2_prev_effect = 'S';
#nonamer1-ORF2
my $AA_nonamer2_ORF2_prev_MUT = get_amino_acid($codon_nonamer2_ORF2_prev_MUT);
if($AA_nonamer2_ORF2_prev_MUT ne $AA_nonamer2_ORF2_prev) {
$nonamer2_ORF2_prev_effect = 'N';
}
if($AA_nonamer2_ORF2_prev eq '*' || $AA_nonamer2_ORF2_prev_MUT eq '*') {
$nonamer2_STOP_caused++;
}
# EASTER: enumerate all possible, site 1
if($easter) { print "$codon_in_seq\tnonamer2\t$codon_nonamer2_ORF1\t$codon_nonamer2_ORF2_prev\t$nt1_nonamer2_WT\t$nt\t$AA_nonamer2_ORF1\t$AA_nonamer2_ORF2_prev\t1\t$AA_nonamer2_ORF1_MUT\t$AA_nonamer2_ORF2_prev_MUT\n" }
# TALLY VIABLE CHANGES, GET NUMBER OF DIFFS
unless($nonamer2_STOP_caused > 0) {
$nonamer2_num_changes_poss_site1++;
if($nonamer2_ORF1_effect eq 'N') {
if($nonamer2_ORF2_prev_effect eq 'N') {
$nonamer2_num_changes_NN_site1++;
} elsif($nonamer2_ORF2_prev_effect eq 'S') {
$nonamer2_num_changes_NS_site1++;
}
} elsif($nonamer2_ORF1_effect eq 'S') {
if($nonamer2_ORF2_prev_effect eq 'N') {
$nonamer2_num_changes_SN_site1++;
} elsif($nonamer2_ORF2_prev_effect eq 'S') {
$nonamer2_num_changes_SS_site1++;
}
}
}
} # end member 2 site 1
# SITE 2
# What is each change to ORF1 CODON SITE 2 / ORF2 CODON SITE 3?
if($nt ne $nt2_nonamer2_WT) { # only one possibility for this site
my $nonamer2_STOP_caused = 0;
my $codon_nonamer2_ORF1_MUT = $codon_nonamer2_ORF1;
$codon_nonamer2_ORF1_MUT =~ s/([ACGT])[ACGT]([ACGT])/$1$nt$2/;
my $codon_nonamer2_ORF2_prev_MUT = $codon_nonamer2_ORF2_prev;
$codon_nonamer2_ORF2_prev_MUT =~ s/([ACGT])([ACGT])[ACGT]/$1$2$nt/;
my $nonamer2_ORF1_effect = 'S';
#nonamer1-ORF1
my $AA_nonamer2_ORF1_MUT = get_amino_acid($codon_nonamer2_ORF1_MUT);
if($AA_nonamer2_ORF1_MUT ne $AA_nonamer2_ORF1) {
$nonamer2_ORF1_effect = 'N';
}
if($AA_nonamer2_ORF1 eq '*' || $AA_nonamer2_ORF1_MUT eq '*') {
$nonamer2_STOP_caused++;
}
my $nonamer2_ORF2_prev_effect = 'S';
#nonamer1-ORF2
my $AA_nonamer2_ORF2_prev_MUT = get_amino_acid($codon_nonamer2_ORF2_prev_MUT);
if($AA_nonamer2_ORF2_prev_MUT ne $AA_nonamer2_ORF2_prev) {
$nonamer2_ORF2_prev_effect = 'N';
}
if($AA_nonamer2_ORF2_prev eq '*' || $AA_nonamer2_ORF2_prev_MUT eq '*') {
$nonamer2_STOP_caused++;
}
# EASTER: enumerate all possible, site 2
if($easter) { print "$codon_in_seq\tnonamer2\t$codon_nonamer2_ORF1\t$codon_nonamer2_ORF2_prev\t$nt2_nonamer2_WT\t$nt\t$AA_nonamer2_ORF1\t$AA_nonamer2_ORF2_prev\t2\t$AA_nonamer2_ORF1_MUT\t$AA_nonamer2_ORF2_prev_MUT\n" }
# TALLY VIABLE CHANGES, GET NUMBER OF DIFFS
unless($nonamer2_STOP_caused > 0) {
$nonamer2_num_changes_poss_site2++;
if($nonamer2_ORF1_effect eq 'N') {
if($nonamer2_ORF2_prev_effect eq 'N') {
$nonamer2_num_changes_NN_site2++;
} elsif($nonamer2_ORF2_prev_effect eq 'S') {
$nonamer2_num_changes_NS_site2++;
}
} elsif($nonamer2_ORF1_effect eq 'S') {
if($nonamer2_ORF2_prev_effect eq 'N') {
$nonamer2_num_changes_SN_site2++;
} elsif($nonamer2_ORF2_prev_effect eq 'S') {
$nonamer2_num_changes_SS_site2++;
}
}
}
} # end member 2 site 2
# SITE 3
# What is each change to ORF1 CODON SITE 3 / ORF2 (next) CODON SITE 1?
if($nt ne $nt3_nonamer2_WT) { # only one possibility for this site
my $nonamer2_STOP_caused = 0;
my $codon_nonamer2_ORF1_MUT = $codon_nonamer2_ORF1;
$codon_nonamer2_ORF1_MUT =~ s/([ACGT])([ACGT])[ACGT]/$1$2$nt/;
my $codon_nonamer2_ORF2_next_MUT = $codon_nonamer2_ORF2_next;
$codon_nonamer2_ORF2_next_MUT =~ s/[ACGT]([ACGT])([ACGT])/$nt$1$2/;
my $nonamer2_ORF1_effect = 'S';
#nonamer2-ORF1
my $AA_nonamer2_ORF1_MUT = get_amino_acid($codon_nonamer2_ORF1_MUT);
if($AA_nonamer2_ORF1_MUT ne $AA_nonamer2_ORF1) {
$nonamer2_ORF1_effect = 'N';
}
if($AA_nonamer2_ORF1 eq '*' || $AA_nonamer2_ORF1_MUT eq '*') {
$nonamer2_STOP_caused++;
}
my $nonamer2_ORF2_next_effect = 'S';
#nonamer2-ORF2
my $AA_nonamer2_ORF2_next_MUT = get_amino_acid($codon_nonamer2_ORF2_next_MUT);
if($AA_nonamer2_ORF2_next_MUT ne $AA_nonamer2_ORF2_next) {
$nonamer2_ORF2_next_effect = 'N';
}
if($AA_nonamer2_ORF2_next eq '*' || $AA_nonamer2_ORF2_next_MUT eq '*') {
$nonamer2_STOP_caused++;
}
# EASTER: enumerate all possible, site 3
if($easter) { print "$codon_in_seq\tnonamer2\t$codon_nonamer2_ORF1\t$codon_nonamer2_ORF2_next\t$nt3_nonamer2_WT\t$nt\t$AA_nonamer2_ORF1\t$AA_nonamer2_ORF2_next\t3\t$AA_nonamer2_ORF1_MUT\t$AA_nonamer2_ORF2_next_MUT\n" }
# TALLY VIABLE CHANGES, GET NUMBER OF DIFFS
unless($nonamer2_STOP_caused > 0) {
$nonamer2_num_changes_poss_site3++;
if($nonamer2_ORF1_effect eq 'N') {
if($nonamer2_ORF2_next_effect eq 'N') {
$nonamer2_num_changes_NN_site3++;
} elsif($nonamer2_ORF2_next_effect eq 'S') {
$nonamer2_num_changes_NS_site3++;
}
} elsif($nonamer2_ORF1_effect eq 'S') {
if($nonamer2_ORF2_next_effect eq 'N') {