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vcf2revcom.pl
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vcf2revcom.pl
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#! /usr/bin/perl
# Creates reverse complement versions of all 3 SNPGenie input files.
# Copyright (C) 2015 Chase W. Nelson
# 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
# CONTACT1: nelsoncw@email.sc.edu
# CONTACT2: cwnelson88@gmail.com
# AFFILIATION1: Austin L. Hughes lab, University of South Carolina (Columbia, SC, USA)
# AFFILIATION2: Wen-Hsiung Li lab, Academia Sinica (Taipei, Taiwan)
# ACKNOWLEDGMENTS: written by C.W.N. with support from a National Science Foundation
# Graduate Research Fellowship (DGE-0929297), a National Science Foundation East Asian
# and Pacific Summer Institutes Fellowship, and a University of South Carolina
# Presidential Fellowship.
use strict;
#use warnings;
use IO::Handle;
if(scalar @ARGV != 3) {
die "\n\n## WARNING: The SNPGenie script vcf2revcom needs exactly 3 ".
"arguments, in this order:\n## (1) A '+' strand FASTA (.fa or .fasta) file containing the ".
"reference sequence against which SNPs were called\n## (2) A '+' strand GTF file ".
"containing both '+' and '–' strand products from the '+' strand point of view; ".
"and\n## (3) A '+' strand SNP report in VCF format.\n\n## For example: ".
"snpgenie-vcf2revcom.pl my_snp_report.vcf my_reference_sequence.fasta my_cds_file.gtf\n\n";
}
my $fasta_file_nm = $ARGV[0];
my $gtf_file_nm = $ARGV[1];
my $curr_snp_report_name = $ARGV[2]; # what we're reading from: the original file
# Generate new file name names
my $new_vcf_file_name;
if($curr_snp_report_name =~/\.vcf/) {
$new_vcf_file_name = $` . "_revcom.txt";
} else {
#$new_vcf_file_name = "vcf_revcom.txt";
die "\nThird argument must be a .vcf file\n\n";
}
if(-e $new_vcf_file_name) {
die "\n## $new_vcf_file_name already exists in this directory; delete before proceeding\n\n";
}
my $revcom_seq = &reverse_complement_from_fasta($fasta_file_nm);
my $seq_length = length($revcom_seq);
print "seq length is $seq_length\n";
my $new_gtf_file_name = &generate_reverse_complement_gtf($gtf_file_nm);
my $new_fasta_file_name = &generate_reverse_complement_fasta($fasta_file_nm);
print "\n## Converting $curr_snp_report_name to reverse complement SNPGenie format...\n";
# Populate a hash with product information for + strand
#my %hh_compl_position_info = &populate_product_information_hh($gtf_file_nm);
#my @curr_compl_products_ordered_by_start = sort { $hh_compl_position_info{$a}->{start} <=> $hh_compl_position_info{$b}->{start} } keys %hh_compl_position_info;
my $newline_char = &detect_newline_char($curr_snp_report_name);
$/ = $newline_char;
my $newline_type;
if($newline_char eq "\r\n") {
$newline_type = "Windows (CRLF, \\r\\n\)";
} elsif($newline_char eq "\r") {
$newline_type = "Mac (CR, \\r\)";
} elsif($newline_char eq "\n") {
$newline_type = "Unix (LF, \\n\)";
}
print "\n## In file $curr_snp_report_name, the newline type is: $newline_type\n";
my $index_chrom;
my $index_pos;
my $index_id;
my $index_ref;
my $index_alt;
my $index_qual;
my $index_filter;
my $index_info;
my $index_format;
my $index_sample;
my $seen_index_chrom;
my $seen_index_pos;
my $seen_index_id;
my $seen_index_ref;
my $seen_index_alt;
my $seen_index_qual;
my $seen_index_filter;
my $seen_index_info;
my $seen_index_format;
my $seen_index_sample;
#CHROM POS ID REF ALT QUAL FILTER INFO FORMAT sample1
my $header_line;
open (ORIGINAL_SNP_REPORT, $curr_snp_report_name);
while (<ORIGINAL_SNP_REPORT>) {
chomp;
if($_ =~ /^#(\w+)/) {
$header_line = $_;
if(!($_ =~/\t/)) {
die "\n\n## WARNING:\n## The SNP Report $curr_snp_report_name is ".
"not TAB-delimited (\\t), or there is only one column.\n\n";
}
last;
}
}
close ORIGINAL_SNP_REPORT;
$header_line =~ s/^#//;
#print "\nHEADER LINE IS: $header_line\n\n";
my @header_arr = split("\t",$header_line);
#print "\nHEADER ARRAY IS: @header_arr\n\n";
# Determine the index of each column
for (my $i=0; $i<scalar(@header_arr); $i++) {
if ($header_arr[$i] =~ /CHROM/) {
$index_chrom = $i;
$seen_index_chrom = 1;
} elsif ($header_arr[$i] =~ /POS/) {
$index_pos = $i;
$seen_index_pos = 1;
} elsif ($header_arr[$i] =~ /ID/) {
$index_id = $i;
$seen_index_id = 1;
} elsif ($header_arr[$i] =~ /REF/) {
$index_ref = $i;
$seen_index_ref = 1;
} elsif ($header_arr[$i] =~ /ALT/) { # Will check the value BEGINS with 'SNP'
$index_alt = $i;
$seen_index_alt = 1;
} elsif ($header_arr[$i] =~ /QUAL/) { # Will check the value BEGINS with 'SNP'
$index_qual = $i;
$seen_index_qual = 1;
} elsif ($header_arr[$i] =~ /FILTER/) {
$index_filter = $i;
$seen_index_filter = 1;
} elsif ($header_arr[$i] =~ /INFO/) {
$index_info = $i;
$seen_index_info = 1;
} elsif ($header_arr[$i] =~ /FORMAT/) {
$index_format = $i;
$seen_index_format = 1;
$index_sample = $i+1; # ASSUME the SAMPLE column follows the FORMAT column
}
}
# DIE if one of the headers have not been seen
if ($seen_index_chrom == 0) {
die "\n\n## WARNING: $curr_snp_report_name does not contain the column header \"CHROM\". SNPGenie terminated\n\n";
} elsif ($seen_index_pos == 0) {
die "\n\n## WARNING: $curr_snp_report_name does not contain the column header \"POS\". SNPGenie terminated\n\n";
} elsif ($seen_index_id == 0) {
die "\n\n## WARNING: $curr_snp_report_name does not contain the column header \"ID\". SNPGenie terminated\n\n";
} elsif ($seen_index_ref == 0) {
die "\n\n## WARNING: $curr_snp_report_name does not contain the column header \"REF\". SNPGenie terminated\n\n";
} elsif ($seen_index_alt == 0) {
die "\n\n## WARNING: $curr_snp_report_name does not contain the column header \"ALT\". SNPGenie terminated\n\n";
} elsif ($seen_index_qual == 0) {
die "\n\n## WARNING: $curr_snp_report_name does not contain the column header \"QUAL\". SNPGenie terminated\n\n";
} elsif ($seen_index_filter == 0) {
die "\n\n## WARNING: $curr_snp_report_name does not contain the column header \"FILTER\". SNPGenie terminated\n\n";
} elsif ($seen_index_info == 0) {
die "\n\n## WARNING: $curr_snp_report_name does not contain the column header \"INFO\". SNPGenie terminated\n\n";
} elsif ($seen_index_format == 0) {
die "\n\n## WARNING: $curr_snp_report_name does not contain the column header \"FORMAT\". SNPGenie terminated\n\n";
}
# Product names are not included directly in the VCF. Thus we must use the GTF file
my @product_names_arr = &get_product_names_vcf($gtf_file_nm);
@product_names_arr = sort(@product_names_arr);
# NEED TO BUILD A HASH WITH keys as ALL PRODUCT POSITIONS IN THE GENOME,
# which we will later convert to minus '-' strand coordinates
# Loop through GTF and store product positions with product names in array
my %positions_with_product_hash;
my %products_hash;
# Must be the NEW GTF FILE, with the new + strand products
#print "\nNew GTF file name is: $new_gtf_file_name\n";
open (GTF_INFILE, $new_gtf_file_name);
while (<GTF_INFILE>) {
chomp;
if($_ =~ /CDS\t\d+\t\d+\t[\.\d+]\t\+/) { # Make sure it's on the + strand
my $product;
if($_ =~ /gene_id \"gene\:([\w\s\.']+)\"/) {
$product = $1;
if ((! exists $products_hash{$product}) && ($product ne '')) {
$products_hash{$product} = 1;
}
} elsif($_ =~ /gene_id \"([\w\s\.']+ [\w\s\.']+)\"/) {
$product = $1;
if ((! exists $products_hash{$product}) && ($product ne '')) {
$products_hash{$product} = 1;
}
} elsif($_ =~ /gene_id \"([\w\s\.']+)\"/) {
$product = $1;
if ((! exists $products_hash{$product}) && ($product ne '')) {
$products_hash{$product} = 1;
}
}
if($product) {
if($_ =~ /CDS\t(\d+)\t(\d+)/) {
my $start = $1;
my $stop = $2;
for(my $i = $start; $i <= $stop; $i++) {
push(@{$positions_with_product_hash{$i}},$product);
}
}
}
}
}
close GTF_INFILE;
# NEEDED?
my @product_names_arr = sort(keys %products_hash);
# Now we want to cycle through the file again in order to build a SNPGenie (CLC style)
# version file for output
open(TEMP_FILE_VCF,">>$new_vcf_file_name\_TEMP");
open (ORIGINAL_SNP_REPORT, $curr_snp_report_name);
while (<ORIGINAL_SNP_REPORT>) {
unless(/^#/) { # lines that begins with "##" or "#" are metadata
chomp;
my @line_arr = split(/\t/,$_);
# ONLY DO THE THING IF THE GENEIOUS TYPE IS "Polymorphism"; not CDS.
my $id = $line_arr[$index_id];
# Save the pure records; the GENEIOUS ORDER is:
# chrom => NC_002516.2
# pos => 154
# id => .
# ref => T
# alt => C
# qual => 222
# filter => PASS
# info => DP=262;VDB=0.266664;RPB=0.201403;AF1=1;AC1=2;DP4=1,0,219,38;MQ=60;FQ=-282;PV4=1,1,1,0.29
# format => GT:PL:GQ
# sample1 => 1/1:255,255,0:99
my $chrom_value = $line_arr[$index_chrom];
my $pos_value = $line_arr[$index_pos];
my $id_value = $line_arr[$index_id];
my $ref_value = $line_arr[$index_ref];
my $alt_value = $line_arr[$index_alt];
my $qual_value = $line_arr[$index_qual];
my $filter_value = $line_arr[$index_filter];
my $info_value = $line_arr[$index_info];
my $format_value = $line_arr[$index_format];
my $sample_value = $line_arr[$index_sample];
#my $sample1_value = $line_arr[$index_sample1];
# Extract the needed values; the CLC ORDER will be:
# file_nm
# ref_pos => 350
# THERE WILL BE NO CDS HERE, because not Geneious
# type => SNV OR MNV OR Insertion OR etc.
# ref => G
# allele => A
# count => 270
# cov => 1313
# freq => 20.56
# over_annot => CDS: gag
# REF_POS
my $ref_pos;
# In case there's a less-than sign
if($line_arr[$index_pos] =~ /\<(\d+)/) {
$ref_pos = $1;
}
$ref_pos = $pos_value;
# TYPE
# Find length, nucleotides involved, etc.
my $reference_nts = $ref_value;
my $variant_nts = $alt_value;
my $reference_length = length($reference_nts);
#my $variant_length = length($variant_nts);
my $is_change = 0;
if($reference_nts ne $variant_nts) {
$is_change = 1;
}
# Change the site numbers to the reverse complement strand
# UPDATE $ref_pos for revcom
my $ref_pos = ($seq_length - $ref_pos + 1);
# DETERMINE CLC type and WHICH PRODUCTS OVERLAP THIS SITE ON THIS STRAND
my $clc_type;
my @this_site_products;
if($reference_length == 1) {
$clc_type = 'SNV';
if(exists $positions_with_product_hash{$ref_pos}) {
@this_site_products = @{$positions_with_product_hash{$ref_pos}};
}
} elsif($reference_length > 1) {
$clc_type = 'MNV';
#print "\nThere is a multi-nucleotide variant at $ref_pos;\n".
# "this is not fully supported for VCF\n\n";
# ELABORATE HERE for sites i+1, i+2, etc.
# Actually, we can add lines to those sites, IF VCF even does MNVs
if(exists $positions_with_product_hash{$ref_pos}) {
@this_site_products = @{$positions_with_product_hash{$ref_pos}};
}
}
my $variant1;
my $variant2;
my $variant3;
# CHANGE nucleotides to complements
$reference_nts =~ tr/ACGT/TGCA/;
$variant_nts =~ tr/ACGT/TGCA/;
# EXTRACT variants
if($variant_nts =~ /(\w+),(\w+),(\w+)/) {
$variant1 = $1;
$variant2 = $2;
$variant3 = $3;
} elsif($variant_nts =~ /(\w+),(\w+)/) {
$variant1 = $1;
$variant2 = $2;
} elsif($variant_nts =~ /\w+/) {
$variant1 = $&;
}
# Check all variants are equal lengths, if exist
my $equal_lengths_variants = 0;
if($variant3) {
if($reference_length == length($variant1) && length($variant1) == length($variant2) &&
length($variant2) == length($variant3)) {
$equal_lengths_variants = 1;
}
} elsif($variant2) {
if($reference_length == length($variant1) && length($variant1) == length($variant2)) {
$equal_lengths_variants = 1;
}
} elsif($reference_length == length($variant1)) {
$equal_lengths_variants = 1;
}
# IF the variant(s) are equal lengths, then we can proceed to procees and
# print to file
my $product_entry = '';
if($equal_lengths_variants) {
# Double check reference nucleotide identity
if(($reference_length == 1) && (substr($revcom_seq,($ref_pos-1),1) ne $reference_nts)) {
print "\nSubstring: ".substr($revcom_seq,($ref_pos-1),1)."\nRef: $reference_nts\n";
die "\n## WARNING: New nucleotide at site $ref_pos does not match reverse ".
"complement. SNPGenie terminatred\n\n";
}
if($variant3) { # THERE ARE THREE VARIANTS -- ADD A FLAG!
if($info_value =~ /NS=(\d+)/) { # We've got a VCF SUMMARIZING INDIVIDUALS
print "\n### FILE TYPE NOT FULLY SUPPORTED###\n";
my $num_samples;
$num_samples = $1;
my $variant_freq1;
my $variant_freq2;
my $variant_freq3;
if($info_value =~ /AF=([\d\.]+),([\d\.]+),([\d\.]+)/) {
$variant_freq1 = $1;
$variant_freq2 = $2;
$variant_freq3 = $3;
}
# COUNTS and PERCENTS
my $ref_freq = (1 - ($variant_freq1 + $variant_freq2 + $variant_freq3));
my $ref_count = ($ref_freq * $num_samples);
my $variant_count1 = ($variant_freq1 * $num_samples);
my $variant_count2 = ($variant_freq2 * $num_samples);
my $variant_count3 = ($variant_freq3 * $num_samples);
my $variant_pct1 = (100 * $variant_freq1);
my $variant_pct2 = (100 * $variant_freq2);
my $variant_pct3 = (100 * $variant_freq3);
$product_entry = '';
if(@this_site_products) {
foreach my $product (@this_site_products) {
$product_entry = 'CDS: ' . $product;
# PRINT 3 LINES TO FILE
if($variant_freq1 > 0) {
my $this_line1 = "$curr_snp_report_name\t$ref_pos\t$clc_type\t$reference_nts\t".
"$variant1\t$variant_count1\t$num_samples\t$variant_pct1\tCDS: $product_entry\n";
print TEMP_FILE_VCF "$this_line1";
}
if($variant_freq2 > 0) {
my $this_line2 = "$curr_snp_report_name\t$ref_pos\t$clc_type\t$reference_nts\t".
"$variant2\t$variant_count2\t$num_samples\t$variant_pct2\tCDS: $product_entry\n";
print TEMP_FILE_VCF "$this_line2";
}
if($variant_freq3 > 0) {
my $this_line3 = "$curr_snp_report_name\t$ref_pos\t$clc_type\t$reference_nts\t".
"$variant3\t$variant_count3\t$num_samples\t$variant_pct3\tCDS: $product_entry\n";
print TEMP_FILE_VCF "$this_line3";
}
}
} else {
# PRINT 3 LINES TO FILE
if($variant_freq1 > 0) {
my $this_line1 = "$curr_snp_report_name\t$ref_pos\t$clc_type\t$reference_nts\t".
"$variant1\t$variant_count1\t$num_samples\t$variant_pct1\tCDS: $product_entry\n";
print TEMP_FILE_VCF "$this_line1";
}
if($variant_freq2 > 0) {
my $this_line2 = "$curr_snp_report_name\t$ref_pos\t$clc_type\t$reference_nts\t".
"$variant2\t$variant_count2\t$num_samples\t$variant_pct2\tCDS: $product_entry\n";
print TEMP_FILE_VCF "$this_line2";
}
if($variant_freq3 > 0) {
my $this_line3 = "$curr_snp_report_name\t$ref_pos\t$clc_type\t$reference_nts\t".
"$variant3\t$variant_count3\t$num_samples\t$variant_pct3\tCDS: $product_entry\n";
print TEMP_FILE_VCF "$this_line3";
}
}
} elsif($info_value =~ /DP4=(\d+),(\d+),(\d+),(\d+)/) { # We've got a VCF of POOL
# Warn
print "\n## File $curr_snp_report_name site $ref_pos has three variants ".
"in a pooled (DP4-tag) VCF file.\n## Variant frequencies have been approximated\n";
# These are high-quality reads, so may be less that the actual coverage
my $fwd_ref_reads = $1;
my $rev_ref_reads = $2;
my $fwd_alt_reads = $3;
my $rev_alt_reads = $4;
# COUNTS and FREQS
my $ref_count = ($fwd_ref_reads + $rev_ref_reads);
my $alt_count = ($fwd_alt_reads + $rev_alt_reads);
my $coverage = ($ref_count + $alt_count);
my $variant_count1 = ($alt_count / 3);
my $variant_count2 = ($alt_count / 3);
my $variant_count3 = ($alt_count / 3);
my $variant_freq1 = ($variant_count1 / $coverage);
my $variant_freq2 = ($variant_count2 / $coverage);
my $variant_freq3 = ($variant_count3 / $coverage);
my $variant_pct1 = (100 * $variant_freq1);
my $variant_pct2 = (100 * $variant_freq2);
my $variant_pct3 = (100 * $variant_freq3);
$product_entry = '';
if(@this_site_products) {
foreach my $product (@this_site_products) {
$product_entry = 'CDS: ' . $product;
# PRINT 3 LINES TO FILE
if($variant_freq1 > 0) {
my $this_line1 = "$curr_snp_report_name\t$ref_pos\t$clc_type\t$reference_nts\t".
"$variant1\t$variant_count1\t$coverage\t$variant_pct1\t$product_entry\n";
print TEMP_FILE_VCF "$this_line1";
}
if($variant_freq2 > 0) {
my $this_line2 = "$curr_snp_report_name\t$ref_pos\t$clc_type\t$reference_nts\t".
"$variant2\t$variant_count2\t$coverage\t$variant_pct2\t$product_entry\n";
print TEMP_FILE_VCF "$this_line2";
}
if($variant_freq3 > 0) {
my $this_line3 = "$curr_snp_report_name\t$ref_pos\t$clc_type\t$reference_nts\t".
"$variant3\t$variant_count3\t$coverage\t$variant_pct3\t$product_entry\n";
print TEMP_FILE_VCF "$this_line3";
}
}
} else {
# PRINT 3 LINES TO FILE
if($variant_freq1 > 0) {
my $this_line1 = "$curr_snp_report_name\t$ref_pos\t$clc_type\t$reference_nts\t".
"$variant1\t$variant_count1\t$coverage\t$variant_pct1\t$product_entry\n";
print TEMP_FILE_VCF "$this_line1";
}
if($variant_freq2 > 0) {
my $this_line2 = "$curr_snp_report_name\t$ref_pos\t$clc_type\t$reference_nts\t".
"$variant2\t$variant_count2\t$coverage\t$variant_pct2\t$product_entry\n";
print TEMP_FILE_VCF "$this_line2";
}
if($variant_freq3 > 0) {
my $this_line3 = "$curr_snp_report_name\t$ref_pos\t$clc_type\t$reference_nts\t".
"$variant3\t$variant_count3\t$coverage\t$variant_pct3\t$product_entry\n";
print TEMP_FILE_VCF "$this_line3";
}
}
} elsif($format_value =~ /AD/) { # We've got a VCF of POOL; we need AD and DP
# Find out how many ":" appear before AD, if any
my $prior_to_AD = $`;
my @colons_prior_to_AD = $prior_to_AD =~ /\:/g;
my $colon_count_before_AD = @colons_prior_to_AD;
my $prior_to_DP;
my @colons_prior_to_DP;
my $colon_count_before_DP;
if($format_value =~ /DP/) {
$prior_to_DP = $`;
@colons_prior_to_DP = $prior_to_DP =~ /\:/g;
$colon_count_before_DP = @colons_prior_to_DP;
} else {
die "\n\n## WARNING: $curr_snp_report_name contains AD but not DP data. SNPGenie terminated\n\n";
}
# EXTRACT the VALUE of AD
my @sample_value_arr = split(/\:/,$sample_value);
my $AD1;
my $AD2;
my $AD3;
my $AD4;
if($sample_value_arr[$colon_count_before_AD] =~ /(\d+)\,(\d+)\,(\d+)\,(\d+)/) { # REF and 2 ALTS
$AD1 = $1;
$AD2 = $2;
$AD3 = $3;
$AD4 = $4;
}
# EXTRACT the VALUE of DP (coverage)
my $DP = $sample_value_arr[$colon_count_before_DP];
# COUNTS and FREQS
my $ref_count = $AD1;
my $variant_count1 = $AD2;
my $variant_count2 = $AD3;
my $variant_count3 = $AD4;
my $coverage = ($AD1+$AD2+$AD3+$AD4);
# Warn if the total reads don't equal the coverage
if ($DP != $coverage) {
print "\n## WARNING: In $curr_snp_report_name site $ref_pos".
",\n## the reads total should ".
"equal the coverage ($DP) but is instead: $coverage.".
"\n## The reads total has been used. Please verify your data.\n";
}
my $variant_freq1 = ($variant_count1 / $coverage);
my $variant_freq2 = ($variant_count2 / $coverage);
my $variant_freq3 = ($variant_count3 / $coverage);
my $variant_pct1 = (100 * $variant_freq1);
my $variant_pct2 = (100 * $variant_freq2);
my $variant_pct3 = (100 * $variant_freq3);
$product_entry = '';
if(@this_site_products) {
foreach my $product (@this_site_products) {
$product_entry = 'CDS: ' . $product;
# PRINT 3 LINES TO FILE
if($variant_freq1 > 0) {
my $this_line1 = "$curr_snp_report_name\t$ref_pos\t$clc_type\t$reference_nts\t".
"$variant1\t$variant_count1\t$coverage\t$variant_pct1\t$product_entry\n";
print TEMP_FILE_VCF "$this_line1";
}
if($variant_freq2 > 0) {
my $this_line2 = "$curr_snp_report_name\t$ref_pos\t$clc_type\t$reference_nts\t".
"$variant2\t$variant_count2\t$coverage\t$variant_pct2\t$product_entry\n";
print TEMP_FILE_VCF "$this_line2";
}
if($variant_freq3 > 0) {
my $this_line3 = "$curr_snp_report_name\t$ref_pos\t$clc_type\t$reference_nts\t".
"$variant3\t$variant_count3\t$coverage\t$variant_pct3\t$product_entry\n";
print TEMP_FILE_VCF "$this_line3";
}
}
} else {
# PRINT 3 LINES TO FILE
if($variant_freq1 > 0) {
my $this_line1 = "$curr_snp_report_name\t$ref_pos\t$clc_type\t$reference_nts\t".
"$variant1\t$variant_count1\t$coverage\t$variant_pct1\t$product_entry\n";
print TEMP_FILE_VCF "$this_line1";
}
if($variant_freq2 > 0) {
my $this_line2 = "$curr_snp_report_name\t$ref_pos\t$clc_type\t$reference_nts\t".
"$variant2\t$variant_count2\t$coverage\t$variant_pct2\t$product_entry\n";
print TEMP_FILE_VCF "$this_line2";
}
if($variant_freq3 > 0) {
my $this_line3 = "$curr_snp_report_name\t$ref_pos\t$clc_type\t$reference_nts\t".
"$variant3\t$variant_count3\t$coverage\t$variant_pct3\t$product_entry\n";
print TEMP_FILE_VCF "$this_line3";
}
}
}
} elsif($variant2) { # THERE ARE TWO VARIANTS -- ADD A FLAG!
if($info_value =~ /NS=(\d+)/) { # We've got a VCF summarizing INDIVIDUALS
print "\n### FILE TYPE NOT FULLY SUPPORTED###\n";
my $num_samples;
$num_samples = $1;
my $variant_freq1;
my $variant_freq2;
if($info_value =~ /AF=([\d\.]+),([\d\.]+)/) {
$variant_freq1 = $1;
$variant_freq2 = $2;
}
# COUNTS and PERCENTS
my $ref_freq = (1 - ($variant_freq1 + $variant_freq2));
my $ref_count = ($ref_freq * $num_samples);
my $variant_count1 = ($variant_freq1 * $num_samples);
my $variant_count2 = ($variant_freq2 * $num_samples);
my $variant_pct1 = (100 * $variant_freq1);
my $variant_pct2 = (100 * $variant_freq2);
$product_entry = '';
if(@this_site_products) {
foreach my $product (@this_site_products) {
$product_entry = 'CDS: ' . $product;
# PRINT 2 LINES TO FILE
if($variant_freq1 > 0) {
my $this_line1 = "$curr_snp_report_name\t$ref_pos\t$clc_type\t$reference_nts\t".
"$variant1\t$variant_count1\t$num_samples\t$variant_pct1\tCDS: $product_entry\n";
print TEMP_FILE_VCF "$this_line1";
}
if($variant_freq2 > 0) {
my $this_line2 = "$curr_snp_report_name\t$ref_pos\t$clc_type\t$reference_nts\t".
"$variant2\t$variant_count2\t$num_samples\t$variant_pct2\tCDS: $product_entry\n";
print TEMP_FILE_VCF "$this_line2";
}
}
} else {
# PRINT 2 LINES TO FILE
if($variant_freq1 > 0) {
my $this_line1 = "$curr_snp_report_name\t$ref_pos\t$clc_type\t$reference_nts\t".
"$variant1\t$variant_count1\t$num_samples\t$variant_pct1\tCDS: $product_entry\n";
print TEMP_FILE_VCF "$this_line1";
}
if($variant_freq2 > 0) {
my $this_line2 = "$curr_snp_report_name\t$ref_pos\t$clc_type\t$reference_nts\t".
"$variant2\t$variant_count2\t$num_samples\t$variant_pct2\tCDS: $product_entry\n";
print TEMP_FILE_VCF "$this_line2";
}
}
} elsif($info_value =~ /DP4=(\d+),(\d+),(\d+),(\d+)/) { # We've got a VCF of POOL
# Warn
print "\n## File $curr_snp_report_name site $ref_pos has two variants ".
"in a pooled (DP4-tag) VCF file.\n## Variant frequencies have been approximated\n";
# These are high-quality reads, so may be less that the actual coverage
my $fwd_ref_reads = $1;
my $rev_ref_reads = $2;
my $fwd_alt_reads = $3;
my $rev_alt_reads = $4;
# COUNTS and FREQS
my $ref_count = ($fwd_ref_reads + $rev_ref_reads);
my $alt_count = ($fwd_alt_reads + $rev_alt_reads);
my $coverage = ($ref_count + $alt_count);
my $variant_count1 = ($alt_count / 2);
my $variant_count2 = ($alt_count / 2);
my $variant_freq1 = ($variant_count1 / $coverage);
my $variant_freq2 = ($variant_count2 / $coverage);
my $variant_pct1 = (100 * $variant_freq1);
my $variant_pct2 = (100 * $variant_freq2);
$product_entry = '';
if(@this_site_products) {
foreach my $product (@this_site_products) {
$product_entry = 'CDS: ' . $product;
# PRINT 2 LINES TO FILE
if($variant_freq1 > 0) {
my $this_line1 = "$curr_snp_report_name\t$ref_pos\t$clc_type\t$reference_nts\t".
"$variant1\t$variant_count1\t$coverage\t$variant_pct1\t$product_entry\n";
print TEMP_FILE_VCF "$this_line1";
}
if($variant_freq2 > 0) {
my $this_line2 = "$curr_snp_report_name\t$ref_pos\t$clc_type\t$reference_nts\t".
"$variant2\t$variant_count2\t$coverage\t$variant_pct2\t$product_entry\n";
print TEMP_FILE_VCF "$this_line2";
}
}
} else {
# PRINT 2 LINES TO FILE
if($variant_freq1 > 0) {
my $this_line1 = "$curr_snp_report_name\t$ref_pos\t$clc_type\t$reference_nts\t".
"$variant1\t$variant_count1\t$coverage\t$variant_pct1\t$product_entry\n";
print TEMP_FILE_VCF "$this_line1";
}
if($variant_freq2 > 0) {
my $this_line2 = "$curr_snp_report_name\t$ref_pos\t$clc_type\t$reference_nts\t".
"$variant2\t$variant_count2\t$coverage\t$variant_pct2\t$product_entry\n";
print TEMP_FILE_VCF "$this_line2";
}
}
} elsif($format_value =~ /AD/) { # We've got a VCF of POOL; we need AD and DP
# Find out how many ":" appear before AD, if any
my $prior_to_AD = $`;
my @colons_prior_to_AD = $prior_to_AD =~ /\:/g;
my $colon_count_before_AD = @colons_prior_to_AD;
#print "\n\nColon count before AD: $colon_count_before_AD\n";
my $prior_to_DP;
my @colons_prior_to_DP;
my $colon_count_before_DP;
if($format_value =~ /DP/) {
$prior_to_DP = $`;
@colons_prior_to_DP = $prior_to_DP =~ /\:/g;
$colon_count_before_DP = @colons_prior_to_DP;
#print "\n\nColon count before DP: $colon_count_before_DP\n";
} else {
die "\n\n## WARNING: $curr_snp_report_name contains AD but not DP data. SNPGenie terminated\n\n";
}
# GENERATE a regex for what comes before the VALUE of AD
#my $prior_to_AD_regex;
#for (my $i=0; $i<$colon_count_before_AD; $i++) {
# $prior_to_AD_regex .= "[\d\w\/\,]+\:";
#}
# EXTRACT the VALUE of AD
my @sample_value_arr = split(/\:/,$sample_value);
my $AD1;
my $AD2;
my $AD3;
if($sample_value_arr[$colon_count_before_AD] =~ /(\d+)\,(\d+)\,(\d+)/) { # REF and 2 ALTS
$AD1 = $1;
$AD2 = $2;
$AD3 = $3;
}
# EXTRACT the VALUE of DP (coverage)
my $DP = $sample_value_arr[$colon_count_before_DP];
# COUNTS and FREQS
my $ref_count = $AD1;
my $variant_count1 = $AD2;
my $variant_count2 = $AD3;
my $coverage = ($AD1+$AD2+$AD3);
# Warn if the total reads don't equal the coverage
if ($DP != $coverage) {
print "\n## WARNING: In $curr_snp_report_name site $ref_pos".
",\n## the reads total should ".
"equal the coverage ($DP) but is instead: $coverage.".
"\n## The reads total has been used. Please verify your data.\n";
}
my $variant_freq1 = ($variant_count1 / $coverage);
my $variant_freq2 = ($variant_count2 / $coverage);
my $variant_pct1 = (100 * $variant_freq1);
my $variant_pct2 = (100 * $variant_freq2);
$product_entry = '';
if(@this_site_products) {
foreach my $product (@this_site_products) {
$product_entry = 'CDS: ' . $product;
# PRINT 2 LINES TO FILE
if($variant_freq1 > 0) {
my $this_line1 = "$curr_snp_report_name\t$ref_pos\t$clc_type\t$reference_nts\t".
"$variant1\t$variant_count1\t$coverage\t$variant_pct1\t$product_entry\n";
print TEMP_FILE_VCF "$this_line1";
}
if($variant_freq2 > 0) {
my $this_line2 = "$curr_snp_report_name\t$ref_pos\t$clc_type\t$reference_nts\t".
"$variant2\t$variant_count2\t$coverage\t$variant_pct2\t$product_entry\n";
print TEMP_FILE_VCF "$this_line2";
}
}
} else {
# PRINT 2 LINES TO FILE
if($variant_freq1 > 0) {
my $this_line1 = "$curr_snp_report_name\t$ref_pos\t$clc_type\t$reference_nts\t".
"$variant1\t$variant_count1\t$coverage\t$variant_pct1\t$product_entry\n";
print TEMP_FILE_VCF "$this_line1";
}
if($variant_freq2 > 0) {
my $this_line2 = "$curr_snp_report_name\t$ref_pos\t$clc_type\t$reference_nts\t".
"$variant2\t$variant_count2\t$coverage\t$variant_pct2\t$product_entry\n";
print TEMP_FILE_VCF "$this_line2";
}
}
}
} elsif($variant1) { # THERE IS ONE VARIANT -- no flag needed
if($info_value =~ /NS=(\d+)/) { # We've got a VCF summarizing INDIVIDUALS
print "\n### FILE TYPE NOT FULLY SUPPORTED###\n";
my $num_samples;
$num_samples = $1;
my $variant_freq1;
if($info_value =~ /AF=([\d\.]+)/) {
$variant_freq1 = $1;
}
# COUNTS and PERCENTS
my $ref_freq = (1 - $variant_freq1);
my $ref_count = ($ref_freq * $num_samples);
my $variant_count1 = ($variant_freq1 * $num_samples);
my $variant_pct1 = (100 * $variant_freq1);
$product_entry = '';
if(@this_site_products) {
foreach my $product (@this_site_products) {
$product_entry = 'CDS: ' . $product;
# PRINT 1 LINE TO FILE
if($variant_freq1 > 0) {
my $this_line1 = "$curr_snp_report_name\t$ref_pos\t$clc_type\t$reference_nts\t".
"$variant1\t$variant_count1\t$num_samples\t$variant_pct1\tCDS: $product_entry\n";
print TEMP_FILE_VCF "$this_line1";
}
}
} else {
# PRINT 1 LINE TO FILE
if($variant_freq1 > 0) {
my $this_line1 = "$curr_snp_report_name\t$ref_pos\t$clc_type\t$reference_nts\t".
"$variant1\t$variant_count1\t$num_samples\t$variant_pct1\tCDS: $product_entry\n";
print TEMP_FILE_VCF "$this_line1";
}
}
} elsif($info_value =~ /DP4=(\d+),(\d+),(\d+),(\d+)/) { # We've got a VCF of POOL
# These are high-quality reads, so may be less that the actual coverage
my $fwd_ref_reads = $1;
my $rev_ref_reads = $2;
my $fwd_alt_reads = $3;
my $rev_alt_reads = $4;
# COUNTS and FREQS
my $ref_count = ($fwd_ref_reads + $rev_ref_reads);
my $alt_count = ($fwd_alt_reads + $rev_alt_reads);
my $coverage = ($ref_count + $alt_count);
my $variant_count1 = $alt_count;
my $variant_freq1 = ($variant_count1 / $coverage);
my $variant_pct1 = (100 * $variant_freq1);
$product_entry = '';
if(@this_site_products) {
foreach my $product (@this_site_products) {
$product_entry = 'CDS: ' . $product;
# PRINT 1 LINE TO FILE
if($variant_freq1 > 0) {
my $this_line1 = "$curr_snp_report_name\t$ref_pos\t$clc_type\t$reference_nts\t".
"$variant1\t$variant_count1\t$coverage\t$variant_pct1\t$product_entry\n";
print TEMP_FILE_VCF "$this_line1";
}
}
} else {
# PRINT 1 LINE TO FILE; $product_entry remains BLANK
if($variant_freq1 > 0) {
my $this_line1 = "$curr_snp_report_name\t$ref_pos\t$clc_type\t$reference_nts\t".
"$variant1\t$variant_count1\t$coverage\t$variant_pct1\t$product_entry\n";
print TEMP_FILE_VCF "$this_line1";
}
}
} elsif($format_value =~ /AD/) { # We've got a VCF of POOL; we need AD and DP
# Find out how many ":" appear before AD, if any
my $prior_to_AD = $`;
my @colons_prior_to_AD = $prior_to_AD =~ /\:/g;
my $colon_count_before_AD = @colons_prior_to_AD;
#print "\n\nColon count before AD: $colon_count_before_AD\n";
my $prior_to_DP;
my @colons_prior_to_DP;
my $colon_count_before_DP;
if($format_value =~ /DP/) {
$prior_to_DP = $`;
@colons_prior_to_DP = $prior_to_DP =~ /\:/g;
$colon_count_before_DP = @colons_prior_to_DP;
#print "\n\nColon count before DP: $colon_count_before_DP\n";
} else {
die "\n\n## WARNING: $curr_snp_report_name contains AD but not DP data. SNPGenie terminated\n\n";
}
# EXTRACT the VALUE of AD
my @sample_value_arr = split(/\:/,$sample_value);
my $AD1;
my $AD2;
if($sample_value_arr[$colon_count_before_AD] =~ /(\d+)\,(\d+)/) { # REF and 1 ALT
$AD1 = $1;
$AD2 = $2;
}
# EXTRACT the VALUE of DP (coverage)
my $DP = $sample_value_arr[$colon_count_before_DP];
# COUNTS and FREQS
my $ref_count = $AD1;
my $variant_count1 = $AD2;
my $coverage = ($AD1+$AD2);
# Warn if the total reads don't equal the coverage
if ($DP != $coverage) {
print "\n## WARNING: In $curr_snp_report_name site $ref_pos".
",\n## the reads total should ".
"equal the coverage ($DP) but is instead: $coverage.".
"\n## The reads total has been used. Please verify your data.\n";
}
my $variant_freq1 = ($variant_count1 / $coverage);
my $variant_pct1 = (100 * $variant_freq1);