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Copy pathIronThrone-GoT
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IronThrone-GoT
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#!/usr/bin/perl
use strict;
use warnings;
use Getopt::Long;
use File::Basename;
use threads;
use List::Util qw(sum);
use Compress::Zlib;
use IO::Uncompress::Gunzip qw(gunzip $GunzipError);
my $what_is_this_code = <<'__conquest_GoT__';
##### created by the Landau lab (@New York Genome Center)
##### for processing GoT amplicon data
__conquest_GoT__
# Global variables, they could be overwritten at init()
my $fastqR1 = undef;
my $fastqR2 = undef;
my $dupcut = undef;
my $my_postP = undef;
my $conf_file = undef;
my $PRIMER_SEQ = undef;
my $PRIMER_ALW = undef;
my $GENERAL_SEQ = undef;
my $GENERAL_ALW = undef;
my $WT_SEQ = undef;
my $WT_ALW = undef;
my $pcr2_fw_wt_SEQ = undef;
my $pcr2_fw_wt_ALW = undef;
my $pcr2_fw_mut_SEQ = undef;
my $pcr2_fw_mut_ALW = undef;
my $pcr2_rv_wt_SEQ = undef;
my $pcr2_rv_wt_ALW = undef;
my $pcr2_rv_mut_SEQ = undef;
my $pcr2_rv_mut_ALW = undef;
my %MATRIX_HEADER;
my @WhitelistBarcodes;
my %WhitelistBarcodes_hash;
my %BARCODE_stringdist_hamm;
##################################################
# Parse paramters then assign provided global variables
##################################################
# default values
my $run_mode = "linear";
my $MMTCH = 0.2 ;
$my_postP = 0.99 ;
$dupcut = 1 ;
my $thread_max = 4;
my $len_barcode = 16;
my $len_umi = 10;
my $ALLOW_ERR = 0;
my $VERBOSE = 0 ;
my $keep_outs = 0 ;
my $out_dir = "./out";
my $samplename = "myGoT";
my $log_file="$samplename.log";
my $dir=dirname($0);
my $whitelist_file="$dir/barcodes10X/737K-august-2016.txt"; # provided from 10x
##################################################
# initiation of pipeline
##################################################
init();
open LOG, ">$out_dir/$log_file" || die $!; close LOG; # clear old file if exists
open LOG, ">>$out_dir/$log_file" || die $!; # open log file for append mode
##################################################
# main function
##################################################
pre_process() ;
post_process() ;
##################################################
# Get option parameters
sub init {
GetOptions (
"run|r=s" => \$run_mode,
"fastqR1|f1=s" => \$fastqR1,
"fastqR2|f2=s" => \$fastqR2,
"config=s" => \$conf_file,
"whitelist=s" => \$whitelist_file,
"mmtch=s" => \$MMTCH,
"postP=s" => \$my_postP,
"dupcut=s" => \$dupcut,
"bclen=s" => \$len_barcode,
"umilen=s" => \$len_umi,
"sample=s" => \$samplename,
"outdir=s" => \$out_dir,
"log=s" => \$log_file,
"thread|t=s" => \$thread_max,
"keepouts=s" => \$keep_outs,
"vervose=s" => \$VERBOSE,
"help" => sub { usage() },
);
if(!defined $fastqR1) {usage(); exit 1 }
if(!defined $fastqR2) {usage(); exit 1 }
if(!defined $conf_file){usage(); exit 1 }
unless(-e $out_dir) {
`mkdir -p $out_dir`;
}
}
# Show option parameters
sub usage {
print <<"USAGE";
$what_is_this_code
USAGE =================================================================================================
IronThrone-GoT [options] --run linear --fastqR1 <in.R1.fastq> --fastqR2 <in.R2.fastq> \\
--config <in.config> --sample <out.prefix> --outdir <out.path>
REQUIRED INPUTS =======================================================================================
-r/--run run module for processing 'linear'/'circ' GoT (default: linear)
-f1/--fastqR1 input R1.FASTQ FILE (input file can be in GZip format with .gz extension)
-f2/--fastqR2 input R2.FASTQ FILE (input file can be in GZip format with .gz extension)
-c/--config input CONFIG FILE (input file should be in tab-separated)
|
| .config file (for 'linear' GoT) should be prepared as follows
| to scan expected sequences at specific positions in amplicon reads
|
| (1st row) primer.sequence start.pos end.pos
| (2nd row) shared.sequence start.pos end.pos
| (3rd row) WT.sequence start.pos end.pos
| (4th row) MUT.sequence start.pos end.pos
|
| as an example, see testdata/linear_example.config
| or README file to prepare circ_example.config
OPTIONS ===============================================================================================
-m/--mmtch allowed mismatch ratio to grep the expected sequences (default: 0.2)
-p/--postP cutoff for the posterior probability in the barcode replacement (default: 0.99)
-d/--dupcut cutoff for the total number of duplication (default: 1)
-b/--bclen length of barcode (default: 16)
-u/--umilen length of UMI (default: 10)
-w/--whitelist file for whitelisted barcodes (737K-august-2016.txt)
-s/--sample prefix for outputs (myGoT)
-o/--outdir path for outputs (./out)
-l/--log logfile name (myGoT.log)
-t/--thread number of threads to run in parallel (default: 4)
-k/--keepouts if set to 1, keeping intermediate files (default: 0)
-v/--verbose if set to 1, returning more logs (default: 0)
-h/--help
USAGE
exit ;
}
##################################################
# Parse necessary information from paired fastq files
sub pre_process {
($fastqR1, $fastqR2) = un_gzip($fastqR1, $fastqR2); # unzip if .gz
sed_repeat($fastqR1, "$out_dir/$samplename.BARCODE", 2, 4, 1, $len_barcode);
sed_repeat($fastqR1, "$out_dir/$samplename.BARCODE_Q", 4, 4, 1, $len_barcode);
sed_repeat($fastqR1, "$out_dir/$samplename.UMI", 2, 4, $len_barcode+1, $len_barcode+$len_umi);
sed_repeat($fastqR2, "$out_dir/$samplename.SEQ", 2, 4);
sed_repeat($fastqR2, "$out_dir/$samplename.SEQ_Q", 4, 4);
convert_phred_BQ("$out_dir/$samplename.BARCODE_Q" , "$out_dir/$samplename.BARCODE_Qnum");
convert_numeric_BQ("$out_dir/$samplename.SEQ_Q", "$out_dir/$samplename.SEQ_Qnum");
sum_of_BQ("$out_dir/$samplename.SEQ_Q");
paste_cols(
"$out_dir/$samplename.BARCODE",
"$out_dir/$samplename.BARCODE_Qnum",
"$out_dir/$samplename.UMI",
"$out_dir/$samplename.SEQ",
"$out_dir/$samplename.SEQ_Qnum",
"$out_dir/$samplename.SEQ_Q.P_errors_SUM",
"$out_dir/$samplename.SEQUENCE",
);
if ($run_mode eq "linear") {
seqs_looking("$out_dir/$samplename.SEQUENCE") ;
} elsif ($run_mode eq "circ") {
seqs_looking2("$out_dir/$samplename.SEQUENCE") ;
}
}
##################################################
# Perform additional processing on parsed amplicon data
sub post_process {
##########################
# load .config file
##########################
if ($run_mode eq "linear") {
open CONF, $conf_file or die "[$conf_file] $!";
my @conf_lines = <CONF>;
close CONF;
for(@conf_lines) { chomp; }
$PRIMER_SEQ=(split "\t", $conf_lines[0])[0];
$PRIMER_ALW=sprintf("%.0f", length($PRIMER_SEQ)*$MMTCH);
$GENERAL_SEQ=(split "\t", $conf_lines[1])[0];
$GENERAL_ALW=sprintf("%.0f", length($GENERAL_SEQ)*$MMTCH);
$WT_SEQ=(split "\t", $conf_lines[2])[0];
$WT_ALW=sprintf("%.0f", length($WT_SEQ)*$MMTCH);
} elsif ($run_mode eq "circ") {
open CONF, $conf_file or die "[$conf_file] $!";
my @conf_lines = <CONF>;
close CONF;
for(@conf_lines) { chomp; }
$PRIMER_SEQ=(split "\t", $conf_lines[0])[0];
$PRIMER_ALW=sprintf("%.0f", length($PRIMER_SEQ)*$MMTCH);
$GENERAL_SEQ=(split "\t", $conf_lines[1])[0];
$GENERAL_ALW=sprintf("%.0f", length($GENERAL_SEQ)*$MMTCH);
$WT_SEQ=(split "\t", $conf_lines[2])[0];
$WT_ALW=sprintf("%.0f", length($WT_SEQ)*$MMTCH);
$pcr2_fw_wt_SEQ=(split "\t", $conf_lines[4])[0];
$pcr2_fw_wt_ALW=sprintf("%.0f", length($pcr2_fw_wt_SEQ)*$ALLOW_ERR);
$pcr2_rv_wt_SEQ=(split "\t", $conf_lines[6])[0];
$pcr2_rv_wt_ALW=sprintf("%.0f", length($pcr2_rv_wt_SEQ)*$ALLOW_ERR);
}
## Filter primed reads by mismatch allowance
my @primed_looked;
my @primed_looked_barcode;
open LOOKED, "$out_dir/$samplename.looked" or die $!;
my @LOOKED_RAW = <LOOKED>;
close LOOKED;
my $file_count_looked = @LOOKED_RAW;
if ($run_mode eq "linear") {
logging("seeking for mismatches from $file_count_looked lines | Primer<= $PRIMER_ALW | Shared<= $GENERAL_ALW ") if $VERBOSE;
for my $line (@LOOKED_RAW) {
my ($barcode, $mm_primer, $mm_share) = (split "\t", $line)[0, 3, 4];
if ($mm_primer <= $PRIMER_ALW && $mm_share <= $GENERAL_ALW) {
push @primed_looked, $line;
push @primed_looked_barcode, $barcode;
}
}
my $primed_looked_barcode_cnt = @primed_looked_barcode;
logging(" -> $primed_looked_barcode_cnt found from $file_count_looked lines") if $VERBOSE;
} elsif ($run_mode eq "circ") {
logging("seeking for mismatches from $file_count_looked lines | Primer<= $PRIMER_ALW | Shared<= $GENERAL_ALW | PCR2FW_WT/PCR2FW_MUT<= $pcr2_fw_wt_ALW | PCR2RV_WT/PCR2RV_MUT<= $pcr2_rv_wt_ALW ") if $VERBOSE;
for my $line (@LOOKED_RAW) {
my ($barcode, $mm_primer, $mm_share, $mm_pcr2fw_wt, $mm_pcr2fw_mut, $mm_pcr2rv_wt, $mm_pcr2rv_mut) = (split "\t", $line)[0, 3, 4, 7, 8, 9, 10];
if ($mm_primer <= $PRIMER_ALW && $mm_share <= $GENERAL_ALW && ($mm_pcr2fw_wt <= $pcr2_fw_wt_ALW || $mm_pcr2fw_mut <= $pcr2_fw_wt_ALW) && ($mm_pcr2rv_wt <= $pcr2_rv_wt_ALW || $mm_pcr2rv_mut <= $pcr2_rv_wt_ALW)) {
push @primed_looked, $line;
push @primed_looked_barcode, $barcode;
}
}
my $primed_looked_barcode_cnt = @primed_looked_barcode;
logging(" -> $primed_looked_barcode_cnt found from $file_count_looked lines") if $VERBOSE;
}
# Load barcodes whitelist
my $arr_tmp = file_read($whitelist_file);
@WhitelistBarcodes = @$arr_tmp;
my @obs_uniq_barcodes = unique(@primed_looked_barcode);
$arr_tmp = intersect(\@WhitelistBarcodes, \@obs_uniq_barcodes);
my @obs_barcodes_in_whitelist = @$arr_tmp;
my @obs_barcodes_NOTin_whitelist = @obs_uniq_barcodes[ find_in_array("not in", \@obs_uniq_barcodes, \@obs_barcodes_in_whitelist) ];
my @MTX_done = array_to_matrix( @primed_looked[ find_in_array("in", \@primed_looked_barcode, \@obs_barcodes_in_whitelist) ] );
my @MTX_ND = array_to_matrix( @primed_looked[ find_in_array("in", \@primed_looked_barcode, \@obs_barcodes_NOTin_whitelist) ] );
matrix_header_create("MTX_done", $MTX_done[0] );
matrix_header_create("MTX_ND", $MTX_ND[0] );
matrix_put_values(\@MTX_done, "MTX_done", "whitelist", "Y");
matrix_put_values(\@MTX_ND, "MTX_ND", "whitelist", "N");
my @MTX_sub = @MTX_ND;
matrix_header_create("MTX_sub", undef, matrix_header_get("MTX_ND" ));
matrix_header_rename("MTX_sub", "V1", "original_BC");
matrix_put_values(\@MTX_sub, "MTX_sub", "replaced_BC", "NA");
stringdist_hamm_pre_calc(\@MTX_sub); # pre-calculate stringdist_hamm() by multi-thread
# Test barcode replacement probability
my @tested;
for (my $r=0; $r < @MTX_sub; $r++) {
my $ori_BC = $MTX_sub[$r][0];
my @BQ = split ";", $MTX_sub[$r][1];
my @Hamm_1 = ();
my $ref = $BARCODE_stringdist_hamm{ $ori_BC };
if ($ref) {
@Hamm_1 = @$ref;
}
if (@Hamm_1 < 1) {
$MTX_sub[$r][ matrix_header_get_index("MTX_sub", "replaced_BC") ] = "No_Hamm_1";
} else {
my %dat_n_i;
my @dat_n;
my @dat_n_header = ("class", "bc", "qv", "At", "readN", "priorP", "p_edit", "likelihood", "postP");
matrix_header_create("dat_n", undef, @dat_n_header );
matrix_header_create("maxdat_n", undef, @dat_n_header );
for (my $i=0; $i < @Hamm_1; $i++) {
my $Hamm_1_i = $Hamm_1[$i];
my $diff_pos = str_diff_pos_1($ori_BC, $Hamm_1_i);
my @tmp_data_test = ($Hamm_1_i);
my @tmp_data_barcode = matrix_get_array_by_col(\@MTX_done, 0);
my @inMTX_done_i = matrix_get_matrix_by_row(\@MTX_done, find_in_array("in", \@tmp_data_barcode, \@tmp_data_test) );
my $NinMTX_done_i = @inMTX_done_i;
if (@inMTX_done_i ==0) {
$MTX_sub[$r][ matrix_header_get_index("MTX_sub", "replaced_BC") ] = "NO_in_SAVED";
my @tmp = ($i, $Hamm_1_i, 0, $diff_pos, 0);
push @dat_n, \@tmp;
} else {
my $Errs_n_i = (split ";", $MTX_sub[$r][1])[$diff_pos];
my $ErrsEdit_n_i = min_value(33, $Errs_n_i);
my @tmp = ($i, $Hamm_1_i, $ErrsEdit_n_i, $diff_pos, $NinMTX_done_i);
push @dat_n, \@tmp;
}
}
matrix_calc_priorP(\@dat_n, "dat_n");
matrix_calc_p_edit(\@dat_n, "dat_n");
matrix_calc_likelihood(\@dat_n, "dat_n");
matrix_calc_postP(\@dat_n, "dat_n");
my @Maxdat_n = matrix_calc_maxdat_n(\@dat_n, "dat_n", "postP");
if ( matrix_sum_cols(\@dat_n, matrix_header_get_index("dat_n", "readN") ) == 0 && @Maxdat_n >= 2) {
@Maxdat_n = ($Maxdat_n[0]);
$MTX_sub[$r][ matrix_header_get_index("MTX_sub", "replaced_BC") ] = "NO_in_SAVED";
}
if ($Maxdat_n[0][ matrix_header_get_index("maxdat_n", "postP") ] ne "NA" && $Maxdat_n[0][ matrix_header_get_index("maxdat_n", "postP") ] >= $my_postP) {
$MTX_sub[$r][ matrix_header_get_index("MTX_sub", "replaced_BC") ] = $Maxdat_n[0][ matrix_header_get_index("maxdat_n", "bc") ];
} else {
$MTX_sub[$r][ matrix_header_get_index("MTX_sub", "replaced_BC") ] = "Not_Sig";
}
if (@Maxdat_n == 2 && $Maxdat_n[0][ matrix_header_get_index("maxdat_n", "postP") ] == 0.5) {
$MTX_sub[$r][ matrix_header_get_index("MTX_sub", "replaced_BC") ] = "Not_Sig";
@Maxdat_n = ($Maxdat_n[0]);
}
}
logging("processing row $r") if $VERBOSE;
push @tested, $MTX_sub[$r];
}
my @MTX_changed = @tested;
matrix_header_create("MTX_changed", undef, matrix_header_get("MTX_sub") );
matrix_save(\@MTX_changed, "$out_dir/$samplename.MTX_changed.txt", "MTX_changed") if $keep_outs;
# Replace barcode and merge with rescued reads
my @MTX_changed_extracted = matrix_calc_mtx_extract(\@MTX_changed);
matrix_header_create("MTX_changed_extracted", undef, matrix_header_get("MTX_sub") );
matrix_calc_MTX_changed_extracted_put_bc(\@MTX_changed_extracted);
my @MTX_RE;
if ($run_mode eq "linear") {
matrix_remove_col(\@MTX_changed_extracted, "MTX_changed_extracted", 13);
matrix_header_rename("MTX_changed_extracted", "original_BC", "V1");
matrix_header_rename("MTX_changed_extracted", "V13", "whitelist");
@MTX_RE = (@MTX_done, @MTX_changed_extracted);
matrix_header_create("MTX_RE", undef, matrix_header_get("MTX_changed_extracted") );
matrix_header_rename("MTX_RE", "V6", "mismatch.WT");
matrix_header_rename("MTX_RE", "V7", "mismatch.MUT");
matrix_header_rename("MTX_RE", "V11", "BQ.in.WT");
matrix_header_rename("MTX_RE", "V12", "BQ.in.MUT");
} elsif ($run_mode eq "circ") {
matrix_remove_col(\@MTX_changed_extracted, "MTX_changed_extracted", 21);
matrix_header_rename("MTX_changed_extracted", "original_BC", "V1");
matrix_header_rename("MTX_changed_extracted", "V21", "whitelist");
@MTX_RE = (@MTX_done, @MTX_changed_extracted);
matrix_header_create("MTX_RE", undef, matrix_header_get("MTX_changed_extracted") );
matrix_header_rename("MTX_RE", "V6", "mismatch.WT");
matrix_header_rename("MTX_RE", "V7", "mismatch.MUT");
matrix_header_rename("MTX_RE", "V15", "BQ.in.WT");
matrix_header_rename("MTX_RE", "V16", "BQ.in.MUT");
}
matrix_header_insert("MTX_RE", "BARCODE_UMI");
matrix_calc_MTX_RE_BARCODE_UMI(\@MTX_RE, "MTX_RE");
my @mtx_re_barcode_umi_list = matrix_get_array_by_col(\@MTX_RE, matrix_header_get_index("MTX_RE", "BARCODE_UMI"));
my @uniq_BC_UMI = unique(@mtx_re_barcode_umi_list); #BARCODE_UMI
my @MTX_RE_dedup;
for (my $d=0; $d <@uniq_BC_UMI; $d++) {
my $uniq_BC_UMI_d = $uniq_BC_UMI[$d];
my @uniq_BC_UMI_d_arr_tmp = ($uniq_BC_UMI_d);
my @MTX_RE_d = @MTX_RE[ find_in_array("in", \@mtx_re_barcode_umi_list, \@uniq_BC_UMI_d_arr_tmp) ];
my $nrow_MTX_RE_d = @MTX_RE_d;
matrix_header_create("MTX_RE_d", undef, matrix_header_get("MTX_RE") );
matrix_put_values(\@MTX_RE_d, "MTX_RE_d", "total_dups", $nrow_MTX_RE_d);
matrix_put_values(\@MTX_RE_d, "MTX_RE_d", "mismatch.WT.call", "");
matrix_put_values(\@MTX_RE_d, "MTX_RE_d", "mismatch.MUT.call", "");
matrix_put_values(\@MTX_RE_d, "MTX_RE_d", "mismatch.call", "amb");
matrix_calc_MTX_RE_d_mismatch_WT_assign(\@MTX_RE_d, "MTX_RE_d");
matrix_calc_MTX_RE_d_mismatch_MUT_assign(\@MTX_RE_d, "MTX_RE_d");
matrix_calc_MTX_RE_d_mismatch_WT_call_assign(\@MTX_RE_d, "MTX_RE_d");
matrix_calc_MTX_RE_d_mismatch_MUT_call_assign(\@MTX_RE_d, "MTX_RE_d");
matrix_calc_MTX_RE_d_mismatch_amb_assign(\@MTX_RE_d, "MTX_RE_d");
matrix_put_values(\@MTX_RE_d, "MTX_RE_d", "WT.inDups", scalar find_in_matrix("in", \@MTX_RE_d, "MTX_RE_d", "mismatch.call", "WT") );
matrix_put_values(\@MTX_RE_d, "MTX_RE_d", "MUT.inDups", scalar find_in_matrix("in", \@MTX_RE_d, "MTX_RE_d", "mismatch.call", "MUT") );
matrix_put_values(\@MTX_RE_d, "MTX_RE_d", "amb.inDups", scalar find_in_matrix("in", \@MTX_RE_d, "MTX_RE_d", "mismatch.call", "amb") );
#1) Select highest frequency read type (i.e. mutant or wild-type) and obtain quality score
my @MTX_wrap_d;
my @MTX_wrap_d_BEST;
matrix_header_create("MTX_wrap_d", undef, matrix_header_get("MTX_RE_d") );
matrix_header_create("MTX_wrap_d_BEST", undef, matrix_header_get("MTX_RE_d") );
if ($MTX_RE_d[0][ matrix_header_get_index("MTX_RE_d", "WT.inDups") ] > $MTX_RE_d[0][ matrix_header_get_index("MTX_RE_d", "MUT.inDups") ] ) {
@MTX_wrap_d = @MTX_RE_d[ find_in_matrix("in", \@MTX_RE_d, "MTX_RE_d", "mismatch.call", "WT") ];
@MTX_wrap_d_BEST = @MTX_RE_d[ find_in_matrix("in", \@MTX_RE_d, "MTX_RE_d", "BQ.in.WT", matrix_stat_col("min", \@MTX_RE_d, matrix_header_get_index("MTX_RE_d", "BQ.in.WT") ) ) ];
if (@MTX_wrap_d_BEST ==1) {
} else {
#2) If the target quality of the remaining reads is equivalent then select the highest quality barcode read
if (@MTX_wrap_d_BEST) {
@MTX_wrap_d_BEST = @MTX_wrap_d_BEST[ matrix_calc_best_quality(\@MTX_wrap_d_BEST, matrix_header_get_index("MTX_wrap_d_BEST", "V2") ) ];
}
}
}
if ($MTX_RE_d[0][ matrix_header_get_index("MTX_RE_d", "WT.inDups") ] < $MTX_RE_d[0][ matrix_header_get_index("MTX_RE_d", "MUT.inDups") ]) {
@MTX_wrap_d = @MTX_RE_d[ find_in_matrix("in", \@MTX_RE_d, "MTX_RE_d", "mismatch.call", "MUT") ];
@MTX_wrap_d_BEST = @MTX_wrap_d[ find_in_matrix("in", \@MTX_wrap_d, "MTX_wrap_d", "BQ.in.WT", matrix_stat_col("min", \@MTX_wrap_d, matrix_header_get_index("MTX_wrap_d", "BQ.in.WT") ) ) ];
if (@MTX_wrap_d_BEST ==1) {
} else {
if (@MTX_wrap_d_BEST) {
@MTX_wrap_d_BEST = @MTX_wrap_d_BEST[ matrix_calc_best_quality(\@MTX_wrap_d_BEST, matrix_header_get_index("MTX_wrap_d_BEST", "V2") ) ];
}
}
}
#3) If both read types have equivalent frequencies, choose the type with the highest target quality score
if ($MTX_RE_d[0][ matrix_header_get_index("MTX_RE_d", "WT.inDups") ] == $MTX_RE_d[0][ matrix_header_get_index("MTX_RE_d", "MUT.inDups") ]) {
@MTX_wrap_d_BEST = @MTX_RE_d[ find_in_matrix("in", \@MTX_RE_d, "MTX_RE_d", "BQ.in.WT", matrix_stat_col("min", \@MTX_RE_d, matrix_header_get_index("MTX_RE_d", "BQ.in.WT") ) ) ];
if (@MTX_wrap_d_BEST ==1) {
} else {
if (@MTX_wrap_d_BEST) {
@MTX_wrap_d_BEST = @MTX_wrap_d_BEST[ matrix_calc_best_quality(\@MTX_wrap_d_BEST, matrix_header_get_index("MTX_wrap_d_BEST", "V2") ) ];
}
}
}
if ($run_mode eq "linear") {
@MTX_wrap_d_BEST = matrix_remove_col(\@MTX_wrap_d_BEST, "MTX_wrap_d_BEST", 16);
@MTX_wrap_d_BEST = matrix_remove_col(\@MTX_wrap_d_BEST, "MTX_wrap_d_BEST", 15);
@MTX_wrap_d_BEST = matrix_remove_col(\@MTX_wrap_d_BEST, "MTX_wrap_d_BEST", 14);
} elsif ($run_mode eq "circ") {
@MTX_wrap_d_BEST = matrix_remove_col(\@MTX_wrap_d_BEST, "MTX_wrap_d_BEST", 25);
@MTX_wrap_d_BEST = matrix_remove_col(\@MTX_wrap_d_BEST, "MTX_wrap_d_BEST", 24);
@MTX_wrap_d_BEST = matrix_remove_col(\@MTX_wrap_d_BEST, "MTX_wrap_d_BEST", 23);
}
if (@MTX_wrap_d_BEST) {
push @MTX_RE_dedup, @MTX_wrap_d_BEST;
} else {
}
my $uniq_BC_UMI_len = @uniq_BC_UMI;
my $d_no = $d + 1;
logging("processing $d_no out of $uniq_BC_UMI_len") if $VERBOSE;
}
matrix_header_create("MTX_RE_dedup", undef, matrix_header_get("MTX_wrap_d_BEST") );
matrix_save(\@MTX_RE_dedup, "$out_dir/$samplename.MTX_RE.dedup.txt","MTX_RE_dedup") if $keep_outs ;
my @MTX_MERGE = @MTX_RE_dedup;
matrix_header_create("MTX_MERGE", undef, matrix_header_get("MTX_RE_dedup") );
if ($run_mode eq "linear") {
@MTX_MERGE = matrix_remove_col(\@MTX_MERGE, "MTX_MERGE", 13);
my @col_select = (1,13,3,15,16,17,14, 8,9,10,11,12, 4,5,6,7);
for my $e (@col_select) { $e--; }
@MTX_MERGE = matrix_get_col(\@MTX_MERGE, "MTX_MERGE", @col_select);
matrix_header_create("MTX_MERGE", undef, ("BC","whitelist",
"UMI","num.WT.in.dups","num.MUT.in.dups","num.amb.in.dups","call.in.dups",
"avg.base_error.R2",
"avg.base_error.primer","avg.base_error.shared","avg.base_error.WT","avg.base_error.MUT",
"mismatch.primer","mismatch.shared","mismatch.WT","mismatch.MUT") );
} elsif ($run_mode eq "circ") {
@MTX_MERGE = matrix_remove_col(\@MTX_MERGE, "MTX_MERGE", 21);
my @col_select = (1,21,3,23,24,25,22, 12,13,14,15,16, 4,5,6,7, 8,9,10,11);
for my $e (@col_select) { $e--; }
@MTX_MERGE = matrix_get_col(\@MTX_MERGE, "MTX_MERGE", @col_select);
matrix_header_create("MTX_MERGE", undef, ("BC","whitelist",
"UMI","num.WT.in.dups","num.MUT.in.dups","num.amb.in.dups","call.in.dups",
"avg.base_error.R2",
"avg.base_error.primer","avg.base_error.shared","avg.base_error.WT","avg.base_error.MUT",
"mismatch.primer","mismatch.shared","mismatch.WT","mismatch.MUT",
"mismatchPCR2FW_WT", "mismatchPCR2FW_MUT", "mismatchPCR2RV_WT", "mismatchPCR2RV_MUT") );
}
matrix_save(\@MTX_MERGE, "$out_dir/$samplename.MTX_MERGE.txt", "MTX_MERGE") if $keep_outs ;
# Summarize accroding to barcode (for each cell)
my @uniq_BC = unique( matrix_get_array_by_col(\@MTX_MERGE, matrix_header_get_index("MTX_MERGE", "BC") ) );
my @summing_u;
my @summing; # final result table;
for (my $u=0; $u < @uniq_BC; $u++) {
my $uniq_BC_u = $uniq_BC[$u] ;
my @tmp_data_test = ($uniq_BC_u);
my @tmp_data_barcode = matrix_get_array_by_col(\@MTX_MERGE, matrix_header_get_index("MTX_MERGE", "BC") );
my @MTX_MERGE_u = matrix_get_matrix_by_row(\@MTX_MERGE, find_in_array("in", \@tmp_data_barcode, \@tmp_data_test) );
matrix_header_create("MTX_MERGE_u", undef, matrix_header_get("MTX_MERGE") );
matrix_put_values(\@MTX_MERGE_u, "MTX_MERGE_u", "mismatch.WT.call", "" );
matrix_put_values(\@MTX_MERGE_u, "MTX_MERGE_u", "mismatch.MUT.call", "" );
matrix_put_values(\@MTX_MERGE_u, "MTX_MERGE_u", "mismatch.call", "" );
matrix_calc_MTX_MERGE_u_WT_assign(\@MTX_MERGE_u, "MTX_MERGE_u");
matrix_calc_MTX_MERGE_u_MUT_assign(\@MTX_MERGE_u, "MTX_MERGE_u");
matrix_calc_MTX_MERGE_u_WT_call_assign(\@MTX_MERGE_u, "MTX_MERGE_u");
matrix_calc_MTX_MERGE_u_MUT_call_assign(\@MTX_MERGE_u, "MTX_MERGE_u");
matrix_calc_MTX_MERGE_u_amb_assign(\@MTX_MERGE_u, "MTX_MERGE_u");
@MTX_MERGE_u = matrix_remove_col(\@MTX_MERGE_u, "MTX_MERGE_u", matrix_header_get_index("MTX_MERGE_u", "mismatch.MUT.call") );
@MTX_MERGE_u = matrix_remove_col(\@MTX_MERGE_u, "MTX_MERGE_u", matrix_header_get_index("MTX_MERGE_u", "mismatch.WT.call") );
my $arr_ref0 = $MTX_MERGE_u[0];
my $arr_ref0_cnt = @$arr_ref0;
my @arr_ref0_tmp = ( ("0") x ($arr_ref0_cnt-1+3) );
@summing_u = (\@arr_ref0_tmp);
my @summing_u_hd = matrix_header_get("MTX_MERGE_u");
@summing_u_hd = (@summing_u_hd[0..$#summing_u_hd - 1]);
@summing_u_hd = (@summing_u_hd, "WT.calls", "MUT.calls", "amb.calls");
matrix_header_create("summing_u", undef, @summing_u_hd );
$summing_u[0][0] = $MTX_MERGE_u[0][0];
my ($cnt_wt, $cnt_mut, $cnt_amb) = matrix_calc_get_mismatch_call(\@MTX_MERGE_u, "MTX_MERGE_u");
matrix_put_values(\@summing_u, "summing_u", "WT.calls", $cnt_wt );
matrix_put_values(\@summing_u, "summing_u", "MUT.calls", $cnt_mut );
matrix_put_values(\@summing_u, "summing_u", "amb.calls", $cnt_amb );
for (my $c =0; $c < matrix_header_get_ncol("MTX_MERGE_u") -1; $c++) {
my $tmp = join ";", matrix_get_array_by_col(\@MTX_MERGE_u, $c);
matrix_put_values(\@summing_u, "summing_u", matrix_header_get_name("summing_u", $c), $tmp);
}
push @summing, @summing_u;
}
# Filter by total number of duplicates
my @final_sum;
for my $line_ref (@summing) {
my @line_col = @$line_ref;
my $dup_WT = sum(split(";", $line_col[3]));
my $dup_MUT= sum(split(";", $line_col[4]));
my $dup_amb= sum(split(";", $line_col[5]));
push @final_sum, $line_ref if($dup_WT + $dup_MUT + $dup_amb >= $dupcut);
}
unless ($keep_outs) {`rm $out_dir/$samplename.*`}
matrix_save(\@final_sum, "$out_dir/$samplename.summTable.txt", "summing_u");
print "______________________________END____________________________\n";
}
###############
# sub-functions
###############
sub find_mm_1 {
my ($ref, $str, $str_exclude) = @_;
my $mm_cnt;
my @out;
for my $e (@$ref) {
next if $e eq $str_exclude;
$mm_cnt = ($str ^ $e) =~ tr/\001-\255//; # XOR
push @out, $e if $mm_cnt == 1;
}
@out;
}
sub sum_of_BQ {
my ($file_in) = @_;
if (!-e $file_in || -s $file_in <= 0) {
logging("$file_in does not exist or 0 byte!") if $VERBOSE;
exit;
}
my %phred_scores = load_phred_scores();
open (FILE, $file_in) or die $!;
my @MY_input =<FILE>;
close FILE;
open OUT, ">$file_in.P_errors_SUM" or die $!;
my $cnt = 1;
for my $line (@MY_input) {
chomp $line;
my $sum = 0;
my @bases = split(//,$line);
for (my $i=0; $i < @bases; $i++){
$sum += $phred_scores{ $bases[$i] }->{'p_err'};
}
print OUT "$sum\n";
$cnt++;
}
close OUT;
}
sub convert_phred_BQ {
my ($file_in, $file_out) = @_;
if (!-e $file_in || -s $file_in <= 0) {
logging("$file_in does not exist or 0 byte!");
exit;
}
my %phred_scores = load_phred_scores();
open OUT, ">$file_out" || die $!;
open FILE, $file_in || die $!;
while(<FILE>){
chomp;
my @base = split(//,$_);
for (my $i=0; $i <= (length($_)-1); $i++){
print OUT $phred_scores{ $base[$i] }->{'q_score'}.";";
}
print OUT "\n";
}
}
sub convert_numeric_BQ {
my ($file_in, $file_out) = @_;
if (!-e $file_in || -s $file_in <= 0) {
logging("$file_in does not exist or 0 byte!");
exit;
}
my %phred_scores = load_phred_scores();
open OUT, ">$file_out" || die $!;
open FILE, $file_in || die $!;
while(<FILE>){
chomp;
my @base = split(//,$_);
for (my $i=0; $i <= (length($_)-1); $i++){
print OUT $phred_scores{ $base[$i] }->{'p_err'}.";";
}
print OUT "\n";
}
logging("[$file_out] created");
}
sub seqs_looking {
my ($file_in) = @_;
if (!-e $file_in || -s $file_in <= 0) {
logging("$file_in does not exist or 0 byte!");
exit;
}
open CONF, $conf_file or die "[$file_in] $!";
my @conf_lines = <CONF>;
close CONF;
for(@conf_lines) { chomp; }
my $PRIMER_SEQ=(split "\t", $conf_lines[0])[0];
my $PRIMER_LEN=length($PRIMER_SEQ);
my $PRIMER_ALW=sprintf("%.0f", $PRIMER_LEN*$ALLOW_ERR);
my $PRIMER_START=(split "\t", $conf_lines[0])[1];
my $PRIMER_END=(split "\t", $conf_lines[0])[2];
my $PRIMER_MMTCH=sprintf("%.0f", $PRIMER_LEN*$MMTCH);
my $GENERAL_SEQ=(split "\t", $conf_lines[1])[0];
my $GENERAL_START=(split "\t", $conf_lines[1])[1];
my $GENERAL_END=(split "\t", $conf_lines[1])[2];
my $GENERAL_ALW=sprintf("%.0f", length($GENERAL_SEQ)*$ALLOW_ERR);
my $WT_SEQ=(split "\t", $conf_lines[2])[0];
my $WT_START=(split "\t", $conf_lines[2])[1];
my $WT_END=(split "\t", $conf_lines[2])[2];
my $WT_ALW=sprintf("%.0f", length($WT_SEQ)*$ALLOW_ERR);
my $MUT_SEQ=(split "\t", $conf_lines[3])[0];
my $MUT_START=(split "\t", $conf_lines[3])[1];
my $MUT_END=(split "\t", $conf_lines[3])[2];
my $MUT_ALW=sprintf("%.0f", length($MUT_SEQ)*$ALLOW_ERR);
##########################
my $SUM_SEQ_ref = file_read($file_in);
my @SUM_SEQ = @$SUM_SEQ_ref;
my $cnt_tmp = 0;
open (my $look_seq, '>', "$out_dir/$samplename.looked");
my $line_cnt = 0;
for (my $i=0; $i < @SUM_SEQ; $i++) {
$line_cnt++;
my $line_raw = $SUM_SEQ[$i];
chomp $line_raw;
next if $line_raw =~ /^\s*$/;
my @line_col = split("\t", $line_raw);
my $line = $line_col[3];
my $PRIMER_FOUND;
my $PRIMER_STARTz;
my $GENERAL_STARTz;
my $GENERAL_ENDz;
my $WT_STARTz;
my $MUT_STARTz;
my $MUT_ENDz;
my $WT_ENDz;
my $PRIMER_ENDz;
$PRIMER_FOUND = "";
for my $loc (0..3) { # consider four shifted locations
my $seq_in_line = substr($line, $PRIMER_START-1+$loc, length($PRIMER_SEQ)+$loc);
my $PRIMER_FOUND_check = mismatch_check($PRIMER_SEQ, $seq_in_line, $PRIMER_ALW);
if ($PRIMER_FOUND_check <= $PRIMER_MMTCH) {
$PRIMER_FOUND = $PRIMER_FOUND_check ;
$GENERAL_STARTz = $GENERAL_START+$loc ; $GENERAL_ENDz = $GENERAL_END+$loc ;
$WT_STARTz = $WT_START+$loc ; $WT_ENDz = $WT_END+$loc ;
$MUT_STARTz = $MUT_START+$loc ; $MUT_ENDz = $MUT_END+$loc ;
last;
}
}
if ($PRIMER_FOUND eq "") {
$PRIMER_FOUND = $PRIMER_LEN;
$GENERAL_STARTz = $GENERAL_START ; $GENERAL_ENDz = $GENERAL_END ;
$WT_STARTz = $WT_START ; $WT_ENDz = $WT_END ;
$MUT_STARTz = $MUT_START ; $MUT_ENDz = $MUT_END ;
}
my $GENERAL_FOUND = mismatch_check($GENERAL_SEQ, substr($line, $GENERAL_STARTz-1, length($GENERAL_SEQ)), 0);
my $WT_FOUND = mismatch_check($WT_SEQ, substr($line, $WT_STARTz-1, length($WT_SEQ)), 0);
my $MUT_FOUND = mismatch_check($MUT_SEQ, substr($line, $MUT_STARTz-1, length($MUT_SEQ)), 0);
if (!$line or !$line_col[5] or !length($GENERAL_SEQ) or
!sum((split(";", $line_col[4]))[$GENERAL_STARTz-1..$GENERAL_ENDz-1])) {
print "line: $line\n";
exit;
}
my $avg_R2_BQ = $line_col[5]/length($line);
my @R2_BQ = split(";", $line_col[4]);
my @BQ_primer = @R2_BQ[1-1..$PRIMER_LEN-1];
my $BQ_primer_avg = sum(@BQ_primer)/$PRIMER_LEN ;
my @BQ_general = @R2_BQ[$GENERAL_STARTz-1..$GENERAL_ENDz-1];
my $BQ_general_avg = sum(@BQ_general)/length($GENERAL_SEQ) ;
my @BQ_WT = @R2_BQ[$WT_STARTz-1..$WT_ENDz-1];
my $BQ_WT_avg = sum(@BQ_WT)/length($WT_SEQ) ;
my @BQ_MUT = @R2_BQ[$MUT_STARTz-1..$MUT_ENDz-1];
my $BQ_MUT_avg = sum(@BQ_MUT)/length($MUT_SEQ) ;
print $look_seq "$line_col[0]\t$line_col[1]\t$line_col[2]\t$PRIMER_FOUND\t$GENERAL_FOUND\t$WT_FOUND\t$MUT_FOUND\t$avg_R2_BQ\t$BQ_primer_avg\t$BQ_general_avg\t$BQ_WT_avg\t$BQ_MUT_avg\n";
}
close $look_seq;
}
sub seqs_looking2 {
my ($file_in) = @_;
if (!-e $file_in || -s $file_in <= 0) {
logging("$file_in does not exist or 0 byte!");
exit;
}
open CONF, $conf_file or die "[$file_in] $!";
my @conf_lines = <CONF>;
close CONF;
for(@conf_lines) { chomp; }
my $PRIMER_SEQ=(split "\t", $conf_lines[0])[0];
my $PRIMER_LEN=length($PRIMER_SEQ);
my $PRIMER_ALW=sprintf("%.0f", $PRIMER_LEN*$ALLOW_ERR);
my $PRIMER_START=(split "\t", $conf_lines[0])[1];
my $PRIMER_END=(split "\t", $conf_lines[0])[2];
my $PRIMER_MMTCH=sprintf("%.0f", $PRIMER_LEN*$MMTCH);
my $GENERAL_SEQ=(split "\t", $conf_lines[1])[0];
my $GENERAL_START=(split "\t", $conf_lines[1])[1];
my $GENERAL_END=(split "\t", $conf_lines[1])[2];
my $GENERAL_ALW=sprintf("%.0f", length($GENERAL_SEQ)*$ALLOW_ERR);
my $WT_SEQ=(split "\t", $conf_lines[2])[0];
my $WT_START=(split "\t", $conf_lines[2])[1];
my $WT_END=(split "\t", $conf_lines[2])[2];
my $WT_ALW=sprintf("%.0f", length($WT_SEQ)*$ALLOW_ERR);
my $MUT_SEQ=(split "\t", $conf_lines[3])[0];
my $MUT_START=(split "\t", $conf_lines[3])[1];
my $MUT_END=(split "\t", $conf_lines[3])[2];
my $MUT_ALW=sprintf("%.0f", length($MUT_SEQ)*$ALLOW_ERR);
my $pcr2_fw_wt_SEQ=(split "\t", $conf_lines[4])[0];
my $pcr2_fw_wt_START=(split "\t", $conf_lines[4])[1];
my $pcr2_fw_wt_END=(split "\t", $conf_lines[4])[2];
my $pcr2_fw_wt_ALW=sprintf("%.0f", length($pcr2_fw_wt_SEQ)*$ALLOW_ERR);
my $pcr2_fw_mut_SEQ=(split "\t", $conf_lines[5])[0];
my $pcr2_fw_mut_START=(split "\t", $conf_lines[5])[1];
my $pcr2_fw_mut_END=(split "\t", $conf_lines[5])[2];
my $pcr2_fw_mut_ALW=sprintf("%.0f", length($pcr2_fw_mut_SEQ)*$ALLOW_ERR);
my $pcr2_rv_wt_SEQ=(split "\t", $conf_lines[6])[0];
my $pcr2_rv_wt_START=(split "\t", $conf_lines[6])[1];
my $pcr2_rv_wt_END=(split "\t", $conf_lines[6])[2];
my $pcr2_rv_wt_ALW=sprintf("%.0f", length($pcr2_rv_wt_SEQ)*$ALLOW_ERR);
my $pcr2_rv_mut_SEQ=(split "\t", $conf_lines[7])[0];
my $pcr2_rv_mut_START=(split "\t", $conf_lines[7])[1];
my $pcr2_rv_mut_END=(split "\t", $conf_lines[7])[2];
my $pcr2_rv_mut_ALW=sprintf("%.0f", length($pcr2_rv_mut_SEQ)*$ALLOW_ERR);
##########################
my $SUM_SEQ_ref = file_read($file_in);
my @SUM_SEQ = @$SUM_SEQ_ref;
my $cnt_tmp = 0;
open (my $look_seq, '>', "$out_dir/$samplename.looked");
my $line_cnt = 0;
for (my $i=0; $i < @SUM_SEQ; $i++) {
$line_cnt++;
my $line_raw = $SUM_SEQ[$i];
chomp $line_raw;
next if $line_raw =~ /^\s*$/;
my @line_col = split("\t", $line_raw);
my $line = $line_col[3];
my $PRIMER_FOUND;
my $PRIMER_STARTz; my $PRIMER_ENDz;
my $GENERAL_STARTz; my $GENERAL_ENDz;
my $WT_STARTz; my $WT_ENDz;
my $MUT_STARTz; my $MUT_ENDz;
my $pcr2_fw_wt_STARTz ; my $pcr2_fw_wt_ENDz;
my $pcr2_fw_mut_STARTz; my $pcr2_fw_mut_ENDz;
my $pcr2_rv_wt_STARTz ; my $pcr2_rv_wt_ENDz;
my $pcr2_rv_mut_STARTz; my $pcr2_rv_mut_ENDz;
$PRIMER_FOUND = "";
for my $loc (0..3) { # Consider four shifted locations in the case of using staggered primer sets
my $seq_in_line = substr($line, $PRIMER_START-1+$loc, length($PRIMER_SEQ)+$loc);
my $PRIMER_FOUND_check = mismatch_check($PRIMER_SEQ, $seq_in_line, $PRIMER_ALW);
if ($PRIMER_FOUND_check <= $PRIMER_MMTCH) {
$PRIMER_FOUND = $PRIMER_FOUND_check ;
$GENERAL_STARTz = $GENERAL_START+$loc ; $GENERAL_ENDz = $GENERAL_END+$loc ;
$WT_STARTz = $WT_START+$loc ; $WT_ENDz = $WT_END+$loc ;
$MUT_STARTz = $MUT_START+$loc ; $MUT_ENDz = $MUT_END+$loc ;
$pcr2_fw_wt_STARTz = $pcr2_fw_wt_START+$loc ; $pcr2_fw_wt_ENDz = $pcr2_fw_wt_END+$loc ;
$pcr2_fw_mut_STARTz = $pcr2_fw_mut_START+$loc ; $pcr2_fw_mut_ENDz = $pcr2_fw_mut_END+$loc ;
$pcr2_rv_wt_STARTz = $pcr2_rv_wt_START+$loc ; $pcr2_rv_wt_ENDz = $pcr2_rv_wt_END+$loc ;
$pcr2_rv_mut_STARTz = $pcr2_rv_mut_START+$loc ; $pcr2_rv_mut_ENDz = $pcr2_rv_mut_END+$loc ;
last;
}
}
if ($PRIMER_FOUND eq "") {
$PRIMER_FOUND = $PRIMER_LEN;
$GENERAL_STARTz = $GENERAL_START ; $GENERAL_ENDz = $GENERAL_END ;
$WT_STARTz = $WT_START ; $WT_ENDz = $WT_END ;
$MUT_STARTz = $MUT_START ; $MUT_ENDz = $MUT_END ;
$pcr2_fw_wt_STARTz = $pcr2_fw_wt_START ; $pcr2_fw_wt_ENDz = $pcr2_fw_wt_END ;
$pcr2_fw_mut_STARTz = $pcr2_fw_mut_START ; $pcr2_fw_mut_ENDz = $pcr2_fw_mut_END ;
$pcr2_rv_wt_STARTz = $pcr2_rv_wt_START ; $pcr2_rv_wt_ENDz = $pcr2_rv_wt_END ;
$pcr2_rv_mut_STARTz = $pcr2_rv_mut_START ; $pcr2_rv_mut_ENDz = $pcr2_rv_mut_END ;
}
my $GENERAL_FOUND = mismatch_check($GENERAL_SEQ, substr($line, $GENERAL_STARTz-1, length($GENERAL_SEQ)), 0);
my $WT_FOUND = mismatch_check($WT_SEQ, substr($line, $WT_STARTz-1, length($WT_SEQ)), 0);
my $MUT_FOUND = mismatch_check($MUT_SEQ, substr($line, $MUT_STARTz-1, length($MUT_SEQ)), 0);
my $pcr2_fw_wt_FOUND = mismatch_check($pcr2_fw_wt_SEQ, substr($line, $pcr2_fw_wt_STARTz-1, length($pcr2_fw_wt_SEQ)), 0);
my $pcr2_fw_mut_FOUND = mismatch_check($pcr2_fw_mut_SEQ, substr($line, $pcr2_fw_mut_STARTz-1, length($pcr2_fw_mut_SEQ)), 0);
my $pcr2_rv_wt_FOUND = mismatch_check($pcr2_rv_wt_SEQ, substr($line, $pcr2_rv_wt_STARTz-1, length($pcr2_rv_wt_SEQ)), 0);
my $pcr2_rv_mut_FOUND = mismatch_check($pcr2_rv_mut_SEQ, substr($line, $pcr2_rv_mut_STARTz-1, length($pcr2_rv_mut_SEQ)), 0);
if (!$line or !$line_col[5] or !length($GENERAL_SEQ) or
!sum((split(";", $line_col[4]))[$GENERAL_STARTz-1..$GENERAL_ENDz-1])) {
print "line: $line\n";
exit;
}
my $avg_R2_BQ = $line_col[5]/length($line);
my @R2_BQ = split(";", $line_col[4]);
my @BQ_primer = @R2_BQ[1-1..$PRIMER_LEN-1];
my $BQ_primer_avg = sum(@BQ_primer)/$PRIMER_LEN ;
my @BQ_general = @R2_BQ[$GENERAL_STARTz-1..$GENERAL_ENDz-1];
my $BQ_general_avg = sum(@BQ_general)/length($GENERAL_SEQ) ;
my @BQ_WT = @R2_BQ[$WT_STARTz-1..$WT_ENDz-1];
my $BQ_WT_avg = sum(@BQ_WT)/length($WT_SEQ) ;
my @BQ_MUT = @R2_BQ[$MUT_STARTz-1..$MUT_ENDz-1];
my $BQ_MUT_avg = sum(@BQ_MUT)/length($MUT_SEQ) ;
my @BQ_pcr2_fw_wt = @R2_BQ[$pcr2_fw_wt_STARTz-1..$pcr2_fw_wt_ENDz-1];
my $BQ_pcr2_fw_wt_avg = sum(@BQ_pcr2_fw_wt)/length($pcr2_fw_wt_SEQ) ;
my @BQ_pcr2_fw_mut = @R2_BQ[$pcr2_fw_mut_STARTz-1..$pcr2_fw_mut_ENDz-1];
my $BQ_pcr2_fw_mut_avg = sum(@BQ_pcr2_fw_mut)/length($pcr2_fw_mut_SEQ) ;
my @BQ_pcr2_rv_wt = @R2_BQ[$pcr2_rv_wt_STARTz-1..$pcr2_rv_wt_ENDz-1];
my $BQ_pcr2_rv_wt_avg = sum(@BQ_pcr2_rv_wt)/length($pcr2_rv_wt_SEQ) ;
my @BQ_pcr2_rv_mut = @R2_BQ[$pcr2_rv_mut_STARTz-1..$pcr2_rv_mut_ENDz-1];
my $BQ_pcr2_rv_mut_avg = sum(@BQ_pcr2_rv_mut)/length($pcr2_rv_mut_SEQ) ;
print $look_seq "$line_col[0]\t$line_col[1]\t$line_col[2]\t$PRIMER_FOUND\t$GENERAL_FOUND\t$WT_FOUND\t$MUT_FOUND\t$pcr2_fw_wt_FOUND\t$pcr2_fw_mut_FOUND\t$pcr2_rv_wt_FOUND\t$pcr2_rv_mut_FOUND\t$avg_R2_BQ\t$BQ_primer_avg\t$BQ_general_avg\t$BQ_WT_avg\t$BQ_MUT_avg\t$BQ_pcr2_fw_wt_avg\t$BQ_pcr2_fw_mut_avg\t$BQ_pcr2_rv_wt_avg\t$BQ_pcr2_rv_wt_avg\n";
}
close $look_seq;
}