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VIcaller.pl
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VIcaller.pl
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#!usr/bin/env perl
#
# Author: Xun Chen
# Email: [email protected]
# Date: 08/05/2020
# Version: v1.1
#
#
############# updates
#
# Version v1.1:
# 02/20/2020 Debug the validate function;
# 04/15/2019 Debug the validate function;
# 01/10/2019 Debug VIcaller using the RNA-seq FASTQ file as input;
# 01/08/2019 Remove the function of installing depedent tools due to the links to get the installers expired;
# 12/02/2018 Debug VIcaller for the use of split reads;
# 06/17/2018 Correct the input data format, now accept input files with any names;
#
# Version v1.0:
# 07/22/2018 Adding the functions of annotating vector sequences;
# 07/22/2018 Adding the functions of annotating cell line contaminations;
#
#
# Questions for helps:
#
# Contact Xun Chen Ph.D. for any questions
use Getopt::Long qw(:config no_ignore_case);
use strict;
use warnings;
use Cwd qw();
#### Parameter variables;
# main
my $function = $ARGV[0];
my $input_sampleID;
my $threads = 16;
my $help;
my $config;
# detect
my $data_type = "WGS";
my $mode = "standard";
my $file_suffix=".fq.gz";
my $sequencing_type="paired-end";
my $length_insertsize=1500;
my $directory=$0;
my $QS_cutoff;
my $repeat;
my $align_back_to_human;
my $build="hg38";
# validate
my $String;
my $GI;
my $Virus;
# calculate
my $File_suffix_bam;
my $Index_sort;
my $Chr;
my $Position;
my $Breakpoint;
my $Number_reads;
##### variables
GetOptions(
"i|input_sampleID=s" => \$input_sampleID,
"h|help" => \$help,
"t|threads=i" => \$threads,
"c|config=s" => \$config,
"f|file_suffix=s" => \$file_suffix,
"d|data_type=s" => \$data_type,
"m|mode=s" => \$mode,
"s|sequencing_type=s" => \$sequencing_type,
"q|QS_cutoff=i" =>\$QS_cutoff,
"r|repeat" => \$repeat,
"a|align_back_to_human" => \$align_back_to_human,
"l|length_insertsize=i" =>\$length_insertsize,
"b|build=s" =>\$build,
"S|String=s" => \$String,
"G|GI=s" => \$GI,
"V|Virus=s" => \$Virus,
"F|File_suffix_bam=s" => \$File_suffix_bam,
"I|Index_sort" => \$Index_sort,
"C|Chr=s" => \$Chr,
"P|Position=i" => \$Position,
"B|Breakpoint=i" => \$Breakpoint,
"N|Number_reads=i" => \$Number_reads,
);
##### time
my @months = qw( Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec );
my @days = qw(Sun Mon Tue Wed Thu Fri Sat Sun);
my ($sec,$min,$hour,$mday,$mon,$year,$wday,$yday,$isdst) = localtime();
print "\n\n\n#############################################################\n";
print "############### \n";
print "############### $hour:$min:$sec, $months[$mon].$mday\n";
print "############### Author: Xun Chen (Xun.Chen\@uvm.edu and xunchen85\@gmail.com)\n";
print "############### \n";
print "#############################################################\n\n\n\n";
print "~~~~~ Begin...\n";
my $input_sampleID2 =$String;
my $candidate_virus=$Virus;
my @groups;
my $ref;
##### default value
if (!defined ($QS_cutoff)) {
$QS_cutoff = 20;
}
##### check if the function is defined
if(!($function)){
prtErr("#Error: please defined the function\n");
prtUsa();
exit;
}
elsif($function ne "detect" && $function ne "calculate" && $function ne "config" && $function ne "validate" & !defined($help)) {
prtErr("#Error: unknown function defined\n");
prtUsa();
exit;
} else {
print "\n# Functions: VIcaller.pl $function..........\n\n";
}
##### check if sampleID provided
if(!defined($input_sampleID) & !defined($help) && $function eq "detect") {
prtErr("#Error: no samples are provided");
prtUsa();
exit;
}
##### show help page
if(defined($help)){
prtUsa();
exit;
}
##### extract the path of the installed VIcaller software
my @directory=split /\//, $directory;
if($#directory>0){
$directory=join("/",@directory[0..$#directory-1]);
$directory=$directory."/";
} else {
$directory="";
}
##### load_config();
my $human_genome;
my $human_genome_tophat;
my $virus_genome;
my $virus_taxonomy;
my $virus_list;
my $vector_db;
my $cell_line;
my $bowtie2_d="";
my $tophat_d="";
my $bwa_d="";
my $samtools_d="";
my $repeatmasker_d="";
my $dust_d="";
my $NGSQCToolkit_d="";
my $fastuniq_d="";
my $SE_MEI_d="";
my $hydra_d="";
my $blat_d="";
my $blastn_d="";
if (defined($config)){
open CINFIG,"$config";
} elsif (-e $directory."VIcaller.config") {
open CINFIG,"${directory}VIcaller.config";
} else {
print "# Error: missing config file, you can either specify one, or create one using function \"config\"\n";
exit;}
while(<CINFIG>){
my @line=split;
if(!(@line)){next;}
if($line[0] eq "\#"){
if($line[1] eq "human_genome"){$human_genome=$line[3];}
elsif($line[1] eq "human_genome_tophat"){$human_genome_tophat=$line[3];}
elsif($line[1] eq "virus_genome"){$virus_genome=$line[3];}
elsif($line[1] eq "virus_taxonomy"){$virus_taxonomy=$line[3];}
elsif($line[1] eq "virus_list"){$virus_list=$line[3];}
elsif($line[1] eq "cell_line"){$cell_line=$line[3];}
elsif($line[1] eq "vector_db"){$vector_db=$line[3];}
elsif($line[1] eq "bowtie2_d"){$bowtie2_d=$line[3];}
elsif($line[1] eq "tophat_d"){$tophat_d=$line[3];}
elsif($line[1] eq "bwa_d"){$bwa_d=$line[3];}
elsif($line[1] eq "samtools_d"){$samtools_d=$line[3];}
elsif($line[1] eq "repeatmasker_d"){$repeatmasker_d=$line[3];}
elsif($line[1] eq "dust_d"){$dust_d=$line[3];}
elsif($line[1] eq "NGSQCToolkit_d"){$NGSQCToolkit_d=$line[3];}
elsif($line[1] eq "fastuniq_d"){$fastuniq_d=$line[3];}
elsif($line[1] eq "SE_MEI_d"){$SE_MEI_d=$line[3];}
elsif($line[1] eq "hydra_d"){$hydra_d=$line[3];}
elsif($line[1] eq "blat_d"){$blat_d=$line[3];}
elsif($line[1] eq "blastn_d"){$blastn_d=$line[3];}
}
}
system ("chmod a+x ${directory}VIcaller.config");
system ("${directory}./VIcaller.config");
my $thread_1=$threads-1;
my $order=0;
##### 1. detect function
if ($function eq "detect"){
check_input();
print "~~~~~ step 1\n";
##### extract supporting reads
if(${file_suffix} =~ "bam"){
$order=1;
convert_bamtofastq(${input_sampleID});
align_to_hg(${input_sampleID}."_h1",".1fq");
$order=2;
convert_bamtofastq(${input_sampleID}."_h1");
system("mv ${input_sampleID}_h1_sm.bam ${input_sampleID}_sm.bam");
system("mv ${input_sampleID}_h1_su.bam ${input_sampleID}_su.bam");
if($mode eq "standard" || $sequencing_type eq "single-end"){
system("gunzip -c ${input_sampleID}_soft.fastq.gz >${input_sampleID}_1soft.fastq");
system("gunzip -c ${input_sampleID}_h1_soft.fastq.gz >>${input_sampleID}_1soft.fastq");
}
if($sequencing_type eq "paired-end"){
system("mv ${input_sampleID}_h1_h1_1.1fq ${input_sampleID}_1.1fq");
system("mv ${input_sampleID}_h1_h1_2.1fq ${input_sampleID}_2.1fq");
} else {
system("mv ${input_sampleID}_h1_h1.1fq ${input_sampleID}.1fq");
}
} else {
$order=1;
align_to_hg(${input_sampleID},${file_suffix});
convert_bamtofastq(${input_sampleID});
if ($data_type eq "RNA-seq") {
$order=2;
align_to_hg(${input_sampleID}."_h1",".1fq");
convert_bamtofastq(${input_sampleID}."_h1");
system("mv ${input_sampleID}_h1_sm.bam ${input_sampleID}_sm.bam");
system("mv ${input_sampleID}_h1_su.bam ${input_sampleID}_su.bam");
if($mode eq "standard" || $sequencing_type eq "single-end"){
system("gunzip -c ${input_sampleID}_soft.fastq.gz >${input_sampleID}_1soft.fastq");
system("gunzip -c ${input_sampleID}_h1_soft.fastq.gz >>${input_sampleID}_1soft.fastq");
}
if($sequencing_type eq "paired-end"){
system("mv ${input_sampleID}_h1_h1_1.1fq ${input_sampleID}_1.1fq");
system("mv ${input_sampleID}_h1_h1_2.1fq ${input_sampleID}_2.1fq");
} else {
system("mv ${input_sampleID}_h1_h1.1fq ${input_sampleID}.1fq");
}
} else {
if($mode eq "standard" || $sequencing_type eq "single-end"){
system("gunzip -c ${input_sampleID}_soft.fastq.gz >${input_sampleID}_1soft.fastq");
}
if($sequencing_type eq "paired-end"){
system("mv ${input_sampleID}_h1_1.1fq ${input_sampleID}_1.1fq");
system("mv ${input_sampleID}_h1_2.1fq ${input_sampleID}_2.1fq");
} else {
system("mv ${input_sampleID}_h1.1fq ${input_sampleID}.1fq");
}
}
}
##### Filter split reads
open SF1, "${input_sampleID}_1soft.fastq";
open SF2, ">${input_sampleID}_1soft.fastq2";
while (my $tmp1=<SF1>) {
if ($tmp1=~"^\@soft") {
my @tmp1=split /\|/, $tmp1;
if ($tmp1[2]%2 >=1) {
print SF2 "$tmp1";
$tmp1=<SF1>;
print SF2 "$tmp1";
$tmp1=<SF1>;
print SF2 "$tmp1";
$tmp1=<SF1>;
print SF2 "$tmp1";
} else {
$tmp1=<SF1>;
$tmp1=<SF1>;
$tmp1=<SF1>;
}
}
}
close SF1;
close SF2;
system ("mv ${input_sampleID}_1soft.fastq2 ${input_sampleID}_1soft.fastq");
print "~~~~~ step 2\n";
##### quality control
if (defined($QS_cutoff) && $sequencing_type eq "paired-end" && $mode eq "standard"){
fastq_qc(${input_sampleID},1);
} elsif (defined($QS_cutoff) && $sequencing_type eq "paired-end" && $mode eq "fast"){
fastq_qc(${input_sampleID},2);
} elsif (defined($QS_cutoff) && $sequencing_type eq "single-end"){
fastq_qc(${input_sampleID},3);
}
if(!defined($QS_cutoff)){ ##### single-end or standard mode without quality control
if($sequencing_type eq "single-end" || $mode eq "standard"){
system ("perl ${directory}Scripts/Soft_clipping_filter.pl -length 20 -file ${input_sampleID}_1soft.fastq -o ${input_sampleID}");
system ("rm ${input_sampleID}_1soft.fastq");
}
}
print "~~~~~ step 3\n";
##### align to viral genomes
if($sequencing_type eq "paired-end"){
system ("${bwa_d}bwa mem -t $threads -k 19 -r 1.5 -c 100000 -m 50 -T 20 -h 10000 -a -Y -M $virus_genome ${input_sampleID}_1.1fq ${input_sampleID}_2.1fq >${input_sampleID}_vsu.sam");
} else{
system ("${bwa_d}bwa mem -t $threads -k 19 -r 1.5 -c 100000 -m 50 -T 20 -h 10000 -a -Y -M $virus_genome ${input_sampleID}.1fq >${input_sampleID}_vsu.sam");
}
system ("${samtools_d}samtools view -bS -@ $thread_1 ${input_sampleID}_vsu.sam >${input_sampleID}_vsu.bam");
system ("${samtools_d}samtools sort -@ $thread_1 ${input_sampleID}_vsu.bam -o ${input_sampleID}_vsu.sort.bam");
system ("${samtools_d}samtools view -@ $thread_1 ${input_sampleID}_vsu.sort.bam >${input_sampleID}_vsu.sort.sam");
system ("rm ${input_sampleID}_vsu.bam");
system ("rm ${input_sampleID}_vsu.sam");
system ("touch ${input_sampleID}_all_breakpoint");
##### split reads
if($sequencing_type eq "single-end" || ($sequencing_type eq "paired-end" && $mode eq "standard")){
system ("${bwa_d}bwa mem -t $threads -k 19 -r 1.5 -c 100000 -m 50 -T 20 -h 10000 -a -Y -M $virus_genome ${input_sampleID}_1sf.fastq >${input_sampleID}_vsoft.sam");
system ("${samtools_d}samtools view -bS -@ $thread_1 ${input_sampleID}_vsoft.sam >${input_sampleID}_vsoft.bam");
system ("${samtools_d}samtools sort -@ $thread_1 ${input_sampleID}_vsoft.bam -o ${input_sampleID}_vsoft_sort.bam");
system ("${samtools_d}samtools view -@ $thread_1 ${input_sampleID}_vsoft_sort.bam >${input_sampleID}_vsoft_sort.sam");
system ("rm ${input_sampleID}_vsoft.bam");
system ("perl ${directory}Scripts/Soft_clipping_transfer.pl -f ${input_sampleID}_vsoft_sort.sam -taxonomy $virus_taxonomy -o ${input_sampleID}_vsoft_breakpoint");
system ("cat ${input_sampleID}_vsoft_breakpoint >>${input_sampleID}_all_breakpoint");
system ("rm ${input_sampleID}_vsoft_breakpoint");
system ("rm ${input_sampleID}_vsoft_sort.sam");
system ("rm ${input_sampleID}_vsoft.sam");
}
print "~~~~~ step 4\n";
if($sequencing_type eq "paired-end"){
create_type();
system ("perl ${directory}Scripts/Break_point_calling.pl -taxonomy $virus_taxonomy -type ${input_sampleID}.type -position ${input_sampleID}_sm.sam -virus ${input_sampleID}_vsu.sort.sam -o ${input_sampleID}");
system ("rm ${input_sampleID}_sm.sam");
system ("cat ${input_sampleID}_breakpoint >>${input_sampleID}_all_breakpoint");
system ("rm ${input_sampleID}_breakpoint");
}
system ("perl ${directory}Scripts/Reads_summary.pl -i ${input_sampleID}_all_breakpoint -o ${input_sampleID}_summary");
system ("rm ${input_sampleID}_all_breakpoint");
system ("perl ${directory}Scripts/Order_by_virus_sequence.pl -i ${input_sampleID}_summary -o ${input_sampleID}.integration");
my $double_length_insertsize=$length_insertsize * 2;
system ("perl ${directory}Scripts/Filtered_single_reads.pl -i ${input_sampleID}.integration -o ${input_sampleID}.integration2 -r $double_length_insertsize");
system ("mv ${input_sampleID}.integration2 ${input_sampleID}.integration");
print "~~~~~ step 5\n";
##### Infection screening
if($mode eq "standard"){
detect_virus();
system ("rm ${input_sampleID}_vsu.sort.sam");
}
print "~~~~~ step 6\n";
##### obtain fuq reads;
system ("perl ${directory}Scripts/Filtered_fastq.pl $input_sampleID");
if ($sequencing_type eq "single-end"){
system ("rm ${input_sampleID}_1.1fuq ${input_sampleID}_2.1fuq");
}
##### reciprocal alignment
if(defined($align_back_to_human)){
reciprocal_alignment();
}
print "~~~~~ step 7\n";
##### repeats
if(defined($repeat)){
filter_repeats();
}
system ("perl ${directory}Scripts/Filtered_integration-HBV_final.pl ${input_sampleID} >${input_sampleID}.21");
system ("perl ${directory}Scripts/Remove_redundancy_split_reads.pl ${input_sampleID}");
system ("rm ${input_sampleID}.21");
system ("mv ${input_sampleID}.2 ${input_sampleID}.3");
print "~~~~~ step 8\n";
##### output
system ("perl ${directory}Scripts/Assign_reads-filter.pl -i ${input_sampleID}.3 -o ${input_sampleID}.f -r $double_length_insertsize -m f");
system ("perl ${directory}Scripts/Result_filtered.pl ${input_sampleID}.f >${input_sampleID}_f");
system ("perl ${directory}Scripts/Result_visual3-3.pl ${input_sampleID}_f ${input_sampleID} 12 80 >${input_sampleID}_f2");
system ("perl ${directory}Scripts/Result_finalize3.pl ${input_sampleID}_f2 >${input_sampleID}_f22");
system ("perl ${directory}Scripts/Results_get.pl ${input_sampleID}_f22 >${input_sampleID}.virus_f");
my $cmd="";
if ($sequencing_type eq "single-end"){
$cmd = q(awk '{if(($15>=50)&&(($39>2 && $41>=30) || ($39==2 && $41>=50)))print$0}');
} else {
$cmd = q(awk '{if(($15>=50)&&($24>0)&&(($39>2 && $41>=30) || ($39==2 && $41>=50)))print$0}');
}
system ("$cmd ${input_sampleID}.virus_f >${input_sampleID}.virus_f2");
system ("perl ${directory}Scripts/Extract_specific_loci_final_visualization.pl ${input_sampleID}_f2 ${input_sampleID}.virus_f2 >${input_sampleID}.visualization");
system ("perl ${directory}Scripts/Fine_mapped_VI.pl ${input_sampleID}.visualization >${input_sampleID}.fine_mapped");
##### align to vector db
if($sequencing_type eq "paired-end"){
system ("${bwa_d}bwa mem -t $threads -k 19 -r 1.5 -c 100000 -m 50 -T 20 -h 10000 -a -Y -M $vector_db ${input_sampleID}_1.1fuq ${input_sampleID}_2.1fuq >${input_sampleID}_vector.sam");
system ("perl ${directory}Scripts/Filter_vector.pl ${input_sampleID} vector");
}
if ($sequencing_type eq "single-end" || $mode eq "standard") {
system ("${bwa_d}bwa mem -t $threads -k 19 -r 1.5 -c 100000 -m 50 -T 20 -h 10000 -a -Y -M $vector_db ${input_sampleID}_1sf.fuq >${input_sampleID}_vector_sf.sam");
system ("perl ${directory}Scripts/Filter_vector.pl ${input_sampleID} vector_sf");
}
my @path_current2=split /\//, ${input_sampleID};
my $path_current2;
if($#path_current2>0){
my $path_current2=join("/",@path_current2[0..$#path_current2-1]);
my $input3=$path_current2[$#path_current2];
}
else {
$path_current2= Cwd::cwd();
$path_current2=$path_current2."/";
}
##### delete files
system ("rm ${input_sampleID}.integration");
system ("rm ${input_sampleID}.fasta");
system ("rm ${input_sampleID}_summary*");
system ("rm ${input_sampleID}.f");
system ("rm ${input_sampleID}_f");
system ("rm ${input_sampleID}_f22");
if(-e ${input_sampleID}."_1sf.others"){
system ("rm ${input_sampleID}_1sf.others");
}
if($sequencing_type eq "single-end" && $mode eq "fast"){
system ("rm ${input_sampleID}.1fq");
system ("rm ${input_sampleID}_vsu.sort.bam");
system ("rm ${input_sampleID}_vsu.sort.sam");
}
##### validation function
} elsif ($function eq "validate"){
if (!(-d ${directory}."Database/GI")) {
mkdir ${directory}."Database/GI/";
}
@groups=("target","vicaller","human");
v_obtain_seq();
v_index_GI();
v_bwa_validate();
v_combine();
v_blat_validate();
v_blastn_validate();
v_convert();
v_summary();
##### calculate function
} elsif ($function eq "calculate"){
my $position1=$Position-10000;
my $position2=$Position+10000;
if (${File_suffix_bam} =~ "fq" || ${File_suffix_bam} =~ "fastq") {
system ("${samtools_d}samtools sort -@ $thread_1 ${input_sampleID}_h.bam -o ${input_sampleID}_s_h.bam");
system ("${samtools_d}samtools index ${input_sampleID}_s_h.bam");
system ("${samtools_d}samtools view ${input_sampleID}_s_h.bam ${Chr}:${position1}-${position2} >${input_sampleID}_${Chr}_${Position}_h.sort.sam");
} elsif (${File_suffix_bam} =~ ".bam" && defined($Index_sort)){
system ("${samtools_d}samtools view ${input_sampleID}${File_suffix_bam} ${Chr}:${position1}-${position2} >${input_sampleID}_${Chr}_${Position}_h.sort.sam");
} elsif (${File_suffix_bam} =~ ".bam" && !defined($Index_sort)) {
system ("${samtools_d}samtools sort -@ $thread_1 ${input_sampleID}${File_suffix_bam} -o ${input_sampleID}_s${File_suffix_bam}");
system ("${samtools_d}samtools index ${input_sampleID}_s${File_suffix_bam}");
system ("${samtools_d}samtools view ${input_sampleID}_s${File_suffix_bam} ${Chr}:${position1}-${position2} >${input_sampleID}_${Chr}_${Position}_h.sort.sam");
}
system ("sort ${input_sampleID}_${Chr}_${Position}_h.sort.sam | uniq > ${input_sampleID}_${Chr}_${Position}_h.sort.sam2");
system ("perl ${directory}Scripts/C_filtered1.pl ${input_sampleID}_${Chr}_${Position}_h.sort.sam2 >${input_sampleID}_${Chr}_${Position}_bp1");
system ("perl ${directory}Scripts/C_filtered2.pl ${input_sampleID}_${Chr}_${Position}_bp1 >${input_sampleID}_${Chr}_${Position}_bp2");
system ("perl ${directory}Scripts/C_filtered3.pl ${input_sampleID}_${Chr}_${Position}_bp2 $Chr $Position >${input_sampleID}_${Chr}_${Position}.bp");
system ("rm ${input_sampleID}_${Chr}_${Position}_bp2 ${input_sampleID}_${Chr}_${Position}_bp1");
system ("rm ${input_sampleID}_${Chr}_${Position}_h.sort.sam ${input_sampleID}_${Chr}_${Position}_h.sort.sam2");
system ("perl ${directory}Scripts/C_allele_fraction.pl ${input_sampleID}_${Chr}_${Position} ${input_sampleID} $Chr $Position $Number_reads $Breakpoint");
}
##### output
##### fine_mapped file
my %output=();
my $output_header="";
my @output_line=();
my $header="Sample_ID VIcaller_mode QC Reciprocal_alignment Candidate_virus GI Chr. Start End No._chimeric_reads No._split_reads Upstream_breakpoint_on_human Downstream_breakpoint_on_human Upstream_breakpoint_on_virus Downstream_breakpoint_on_virus Information_of_both_upstream_and_downstream_breakpoints Integration_site_in_the_human_genome Integration_allele_fraction No._reads_supporting_nonVI No._reads_supporting_VI Average_alignment_score Is_cell_line_contamination Is_vector Validation_chimeric_confident Validation_chimeric_weak Validation_chimeric_false Validation_split_confident Validation_split_weak Validation_split_false\n";
if ($function eq "detect"){
print "~~~~~ step 9 output of VIcaller detect function\n";
##### open file_mapped
open FINE, "${input_sampleID}.fine_mapped";
while (<FINE>){
@output_line=split;
$output_header=join ("_",(@output_line[0..3],$output_line[5]));
@{$output{$output_header}} = ("-") x 29;
$output{$output_header}[0]=$output_line[0];
if($mode eq "standard"){
$output{$output_header}[1] = "standard";
} elsif ($mode eq "fast"){
$output{$output_header}[1] = "fast";
}
if(defined($QS_cutoff)){
$output{$output_header}[2] = "yes";
} else {
$output{$output_header}[2] = "no";
}
if(defined($align_back_to_human)){
$output{$output_header}[3] = "yes";
} else {
$output{$output_header}[3] = "no";
}
@{$output{$output_header}}[4..5]=@output_line[4..5];
@{$output{$output_header}}[6..8]=@output_line[1..3];
if(($output_line[8] ne "-" && $output_line[17] eq "-") || ($output_line[8] ne "-" && $output_line[17] ne "-" && $output_line[16]<=$output_line[7])){
if ($output_line[7] eq "-") {
$output{$output_header}[15]="E(++);";
$output{$output_header}[11]=$output_line[9];
$output{$output_header}[14]=$output_line[12];
} else {
$output{$output_header}[15]="D(++);";
$output{$output_header}[11]=$output_line[7];
$output{$output_header}[14]=$output_line[10];
}
} elsif (($output_line[8] eq "-" && $output_line[17] ne "-") || ($output_line[8] ne "-" && $output_line[17] ne "-" && $output_line[16]>=$output_line[7])){
if ($output_line[16] eq "-") {
$output{$output_header}[15]="E(+-);";
$output{$output_header}[11]=$output_line[18];
$output{$output_header}[13]=$output_line[20];
} else {
$output{$output_header}[15]="D(+-);";
$output{$output_header}[11]=$output_line[16];
$output{$output_header}[13]=$output_line[19];
}
} elsif ($output_line[8] eq "-" && $output_line[17] eq "-") {
$output{$output_header}[15]="na;";
}
if(($output_line[26] ne "-" && $output_line[35] eq "-") || ($output_line[26] ne "-" && $output_line[35] ne "-" && $output_line[34]<=$output_line[25])){
if ($output_line[25] eq "-") {
$output{$output_header}[15]=$output{$output_header}[15]."e(-+)";
$output{$output_header}[12]=$output_line[26];
$output{$output_header}[14]=$output_line[30];
} else {
$output{$output_header}[15]=$output{$output_header}[15]."d(-+)";
$output{$output_header}[12]=$output_line[25];
$output{$output_header}[14]=$output_line[28];
}
} elsif (($output_line[26] eq "-" && $output_line[35] ne "-") || ($output_line[26] ne "-" && $output_line[35] ne "-" && $output_line[34]>=$output_line[25])){
if ($output_line[34] eq "-") {
$output{$output_header}[15]=$output{$output_header}[15]."e(--)";
$output{$output_header}[12]=$output_line[35];
$output{$output_header}[13]=$output_line[38];
} else {
$output{$output_header}[15]=$output{$output_header}[15]."d(--)";
$output{$output_header}[12]=$output_line[34];
$output{$output_header}[13]=$output_line[37];
}
} elsif ($output_line[26] eq "-" && $output_line[35] eq "-") {
$output{$output_header}[15]=$output{$output_header}[15]."na";
}
if ($output{$output_header}[11] ne "-" && $output{$output_header}[12] ne "-"){
if (($output{$output_header}[15] =~ "D" && $output{$output_header}[15] =~ "d") || ($output{$output_header}[15] =~ "E" && $output{$output_header}[15] =~ "e")){
$output{$output_header}[16]=int(($output{$output_header}[12]+$output{$output_header}[11])/2);
} elsif ($output{$output_header}[15] =~ "D"){
$output{$output_header}[16]=$output{$output_header}[11];
} elsif ($output{$output_header}[15] =~ "d") {
$output{$output_header}[16]=$output{$output_header}[12];
}
} elsif ($output{$output_header}[11] ne "-" && $output{$output_header}[12] eq "-") {
$output{$output_header}[16]=$output{$output_header}[11];
} elsif ($output{$output_header}[12] ne "-" && $output{$output_header}[11] eq "-") {
$output{$output_header}[16]=$output{$output_header}[12];
}
$output{$output_header}[15] =~ s/e/E/;
$output{$output_header}[15] =~ s/d/D/;
##### read cell line
my %cell_line=();
my @cell_line=();
my $col_ID=0;
if ($build eq "hg19"){
$col_ID=3;
} else {
$col_ID=4;
}
open CELL,"$cell_line";
while(<CELL>){
@cell_line=split;
my $cell1=$cell_line[$col_ID];
$cell1=~s/\:/ /;
$cell1=~s/\-/ /;
my @cell1=split /\s+/, $cell1;
if ($output{$output_header}[6] =~ "Chr"){
$cell1[0] =~ s/chr/Chr/;
} elsif ($output{$output_header}[6] =~ "chr") {
$cell1[0] =~ s/chr/chr/;
} elsif (!($output{$output_header}[6] =~ "chr") && !($output{$output_header}[6] =~ "Chr")) {
$cell1[0] =~ s/chr//;
}
@{$cell_line{$cell_line[$col_ID]}}=(@cell_line[0..2],@cell1);
}
close CELL;
my @key_cell1 = keys %cell_line;
my $Is_cell="-";
for (my $ci=0;$ci<@key_cell1;$ci++){
my @cell2 = @{$cell_line{$key_cell1[$ci]}};
if ($cell2[3] eq $output{$output_header}[6]){
if (abs($cell2[4] - $output{$output_header}[7])<=1000 || abs($cell2[4] - $output{$output_header}[8])<=1000 || ($cell2[4]>=$output{$output_header}[7] && $cell2[4]<=$output{$output_header}[8])){ $output{$output_header}[21]="Yes";}
if ($cell2[5] ne "N" && $cell2[5] ne "NA"){
if(abs($cell2[5] - $output{$output_header}[7])<=1000 || abs($cell2[5] - $output{$output_header}[8])<=1000 || ($cell2[5]>=$output{$output_header}[7] && $cell2[5]<=$output{$output_header}[8])){$output{$output_header}[21]="Yes";}
}
}
}
}
##### open vector file
my %vec_line=();
my @vec_line=();
my %v_chimeric=();
my %v_split=();
system ("cat ${input_sampleID}_vector.hmap ${input_sampleID}_vector_sf.hmap > ${input_sampleID}_vector.hmapped");
open VEC, "${input_sampleID}_vector.hmapped";
while (<VEC>){
@vec_line=split;
if (exists ($vec_line{$vec_line[1]})){
if ($vec_line{$vec_line[1]} eq "P") {
next;
} elsif (($vec_line{$vec_line[1]} eq "L" && $vec_line[0] eq "L") || ($vec_line{$vec_line[1]} eq "R" && $vec_line[0] eq "R")) {
next;
} elsif ($vec_line{$vec_line[1]} eq "L" && $vec_line[0] eq "R" && (($vec_line[7]-$vec_line[6]+1) >= 50 || (($vec_line[7]-$vec_line[6]+1)>=30 && ($vec_line[7]-$vec_line[6]+1)/$vec_line[8]>=0.5))) {
$vec_line{$vec_line[1]}="P";
} elsif ($vec_line{$vec_line[1]} eq "R" && $vec_line[0] eq "L" && (($vec_line[4]-$vec_line[3]+1) >= 50 || (($vec_line[4]-$vec_line[3]+1)>=30 && ($vec_line[4]-$vec_line[3]+1)/$vec_line[5]>=0.5))) {
$vec_line{$vec_line[1]}="P";
}
} else {
if (($vec_line[7]-$vec_line[6]+1) >= 50 || ($vec_line[7]-$vec_line[6]+1) eq $vec_line[8] || (($vec_line[7]-$vec_line[6]+1)>=30 && ($vec_line[7]-$vec_line[6]+1)/$vec_line[8]>=0.5)) {
$vec_line{$vec_line[1]}="R";
}
if (($vec_line[4]-$vec_line[3]+1) >= 50 || ($vec_line[4]-$vec_line[3]+1) eq $vec_line[5] || (($vec_line[4]-$vec_line[3]+1)>=30 && ($vec_line[4]-$vec_line[3]+1)/$vec_line[5]>=0.5)) {
if (exists ($vec_line{$vec_line[1]})){
$vec_line{$vec_line[1]}="P";
} else {
$vec_line{$vec_line[1]}="L";
}
}
}
}
##### read virus_f2 file
my @f2_line=();
my $f2_header="";
open F2,"${input_sampleID}.virus_f2";
while (my $f2_line=<F2>){
@f2_line=split /\s+/, $f2_line;
##### check vector
$f2_line[0]=~s/ad://;
my $f2_header2 = $f2_line[0]."_".$f2_line[15]."_".$f2_line[35]."_".$f2_line[30]."_".$f2_line[31]."_".$f2_line[32]."_".$f2_line[33];
my %f2_header2=();
$f2_header2{$f2_header2}=0;
open VIS,"${input_sampleID}.visualization";
while (my $v_line_temp1=<VIS>){
my @f2_title_O1=();
my $f2_title3="";
my $v_ok=0;
my @v_line=split /\s+/, $v_line_temp1;
##### print "VIS: @v_line\n";
unless (@v_line) {next;}
if($v_line[0] eq "O1"){
$v_ok=0;
my $v_title2=$v_line[2]."_".$v_line[1]."_".$v_line[22]."_".$v_line[17]."_".$v_line[18]."_".$v_line[19]."_".$v_line[20];
$f2_title3="";
$v_line[21]= lc $v_line[21];
if($v_line[21] =~ "unknown") {
$v_line[21]=$v_line[22];
}
if (exists ($f2_header2{$v_title2})) {
$v_ok=1;
@f2_title_O1=();
@f2_title_O1=@v_line;
$f2_title3=$v_line[1]." ".$v_line[18]." ".$v_line[19]." ".$v_line[20]." ".$f2_header2{$v_title2}." ".$v_line[22];
}
} elsif ($v_line[0] eq "O2" && $v_ok==1) {
if (exists ($vec_line{$v_line[1]})){
if ($v_line[4] eq "S1"){
if (exists ($v_split{$v_line[0]})){
$v_split{$v_line[0]}="Vector";
} else {
$v_split{$v_line[0]}="Vector";
}
} elsif ($v_line[4] eq "S") {
if ($vec_line{$v_line[1]} eq "P" || $vec_line{$v_line[1]} eq "R") {
$v_chimeric{$v_line[0]}="Vector";
} else {
$v_chimeric{$v_line[0]}="Non-vector";
}
} elsif ($v_line[4] eq "F") {
if ($vec_line{$v_line[1]} eq "P" || $vec_line{$v_line[1]} eq "L") {
$v_chimeric{$v_line[0]}="Vector";
} else {
$v_chimeric{$v_line[0]}="Non-vector";
}
}
} else {
if ($v_line[4] eq "S1") {
$v_split{$v_line[0]}="Non-vector";
} else {
$v_chimeric{$v_line[0]}="Non-vector";
}
}
}
}
close VIS;
##### vector organize
my @key_chimeric=();
my @key_split=();
my $v_chimeric_count=0;
my $v_split_count=0;
for(my $kc=0;$kc<@key_chimeric;$kc++){
if ($v_chimeric{$key_chimeric[$kc]} eq "Vector"){$v_chimeric_count++;}
}
for (my $ks=0;$ks<@key_split;$ks++){
if ($v_split{$key_split[$ks]} eq "Vector"){$v_split_count++;}
}
##### organize;
$f2_header=join ("_",($f2_line[15],@f2_line[31..33],$f2_line[35]));
if(exists($output{$f2_header})){
$output{$f2_header}[9]=$#key_chimeric+1;
$output{$f2_header}[10]=$#key_split+1;
$output{$f2_header}[19]=$f2_line[38];
$output{$f2_header}[20]=$f2_line[40];
if (%v_chimeric && %v_split) {
if ($v_chimeric_count eq ($#key_chimeric+1) && $v_split_count eq ($#key_split+1)) {
$output{$f2_header}[22] = "Yes";
}
} elsif (%v_chimeric) {
if ($v_chimeric_count eq ($#key_chimeric+1)){
$output{$f2_header}[22] = "Yes";}
} elsif (%v_split) {
if ($v_split_count eq ($#key_split+1)) {
$output{$f2_header}[22] = "Yes";
}
} else {
$output{$f2_header}[22] = "-";
}
}
}
##### output
my @list=sort{$output{$b}->[19]<=>$output{$a}->[19]} keys %output;
open OUT, ">${input_sampleID}.output";
print OUT "$header";
$" = "\t";
for(my $i=0;$i<@list;$i++){
print OUT "@{$output{$list[$i]}}\n";
}
close OUT;
}
##### read validation file
my $cs3_header="";
my @cs3_line=();
%output=();
my @out_line="";
my $out_header="";
if ($function eq "validate" && -e ${input_sampleID}.".output") {
print "~~~~~ step 9 output of VIcaller validate function\n";
open OUT, "${input_sampleID}.output";
my $out_temp=<OUT>;
while (<OUT>){
@out_line=split;
$out_header=join ("_",($out_line[0],@out_line[6..8],$out_line[5]));
@{$output{$out_header}}=@out_line;
}
open CS3, "${String}.CS3";
while (<CS3>){
@cs3_line=split;
$cs3_header=join ("_",($cs3_line[57],$cs3_line[58],$cs3_line[59],$cs3_line[60],$cs3_line[62]));
if(exists($output{$cs3_header})){
@{$output{$cs3_header}}[23..25]=@cs3_line[2..4];
@{$output{$cs3_header}}[26..28]=@cs3_line[6..8];
}
}
my @list=sort{$output{$b}->[19]<=>$output{$a}->[19]} keys %output;
open OUT, ">${input_sampleID}.output";
print OUT "$header";
$" = "\t";
for(my $i=0;$i<@list;$i++){
print OUT "@{$output{$list[$i]}}\n";
}
close OUT;
}
##### read bp2 file
if ($function eq "calculate" && -e ${input_sampleID}."_".${Chr}."_".${Position}.".bp2"){
print "~~~~~ step 9 output of VIcaller calculate function\n";
my @out_line="";
my $out_header="";
my @bp2_line=();
%output=();
open BP2,"${input_sampleID}_${Chr}_${Position}.bp2";
my $bp2_line=<BP2>;
$bp2_line=<BP2>;
@bp2_line= split /\s+/, $bp2_line;
if (-e ${input_sampleID}.".output") {
open OUT, "${input_sampleID}.output";
my $out_temp=<OUT>;
while (<OUT>){
@out_line=split;
$out_header=$out_line[0]."_".$out_line[6]."_".$out_line[16];
@{$output{$out_header}}=@out_line;
}
close OUT;
open OUT,">${input_sampleID}.output";
print OUT "$header";
$" = "\t";
my @list=sort{$output{$b}->[19]<=>$output{$a}->[19]} keys %output;
for(my $i=0;$i<@list;$i++){
if ($list[$i] eq $bp2_line[0]){
${$output{$list[$i]}}[17]=$bp2_line[3];
${$output{$list[$i]}}[18]=$bp2_line[1];
}
print OUT "@{$output{$list[$i]}}\n";
}
close OUT;
}
}
##### get local time
@months = qw( Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec );
@days = qw(Sun Mon Tue Wed Thu Fri Sat Sun);
($sec,$min,$hour,$mday,$mon,$year,$wday,$yday,$isdst) = localtime();
print "\n\n\n~~~~~ Done\n#############################################################\n";
print "############### \n";
print "############### $hour:$min:$sec, $months[$mon].$mday\n";
print "############### Author: Xun Chen (Xun.Chen\@uvm.edu and xunchen85\@gmail.com)\n";
print "############### \n";
print "#############################################################\n\n\n\n";
##### all sub functions
##### detect function
##### detect virus
sub detect_virus {
system("perl ${directory}Scripts/Infection_filtered_detection-1-092717.pl ${input_sampleID}_vsu.sort.sam ${input_sampleID}.type 30 >${input_sampleID}_In_infection2");
system("perl ${directory}Scripts/Infection_filtered_detection-2-092717.pl ${input_sampleID}_In_infection2 $virus_list >${input_sampleID}_In_infection22");
system("perl ${directory}Scripts/Infection_filtered_detection-3-092717.pl ${input_sampleID}_In_infection22 >${input_sampleID}_In.virus_v");
system("perl ${directory}Scripts/Infection_filtered_detection-4-092717.pl ${input_sampleID}_In_infection22 >${input_sampleID}_In.virus_g");
system("perl ${directory}Scripts/Infection_Summary_virus-1-092717.pl ${input_sampleID}_In.virus_v ${input_sampleID}_In.virus_g $virus_taxonomy $virus_list >${input_sampleID}_In.virus_v2");
system("perl ${directory}Scripts/Infection_Summary_virus-2-092717.pl ${input_sampleID}_In.virus_v2 5 >${input_sampleID}_In.virus_v3");
if (-e ${input_sampleID}."_In_infection2") {system("rm ${input_sampleID}_In_infection2");}
if (-e ${input_sampleID}."_In_infection22") {system("rm ${input_sampleID}_In_infection22");}
if (-e ${input_sampleID}."_In.virus_v") {system("rm ${input_sampleID}_In.virus_v");}
if (-e ${input_sampleID}."_In.virus_g") {system("rm ${input_sampleID}_In.virus_g");}
if (-e ${input_sampleID}."_In.virus_v2") {system("rm ${input_sampleID}_In.virus_v2");}
}
##### reciprocal alignment
sub reciprocal_alignment {
if($sequencing_type eq "paired-end"){
system ("${bwa_d}bwa mem -t $threads -k 19 -r 1.5 -c 100000 -m 50 -T 20 -h 10000 -a -Y -M $human_genome ${input_sampleID}_1.1fuq ${input_sampleID}_2.1fuq >${input_sampleID}_hpe.sam");
system ("perl ${directory}Scripts/Realign_filter2.pl $input_sampleID");
system ("sort -k 2 -k 3 -k 4 -k 5 -k 6 -k 7 -k 8 -k 9 -k 10 ${input_sampleID}.hmap |uniq >${input_sampleID}.hmapped");
if (-e ${input_sampleID}.".hmap") {system ("rm ${input_sampleID}.hmap");}
if (-e ${input_sampleID}.".hpe.sam") {system ("rm ${input_sampleID}_hpe.sam");}
}
}
##### check repeat
sub filter_repeats {
my $path_current= Cwd::cwd();
my @directory1=split /\//, ${input_sampleID};
if($#directory1>0){
my $directory1=join("/",@directory1[0..$#directory1-1]);
my $input2=$directory1[$#directory1];
print "$path_current\n";
chdir $path_current."/".$directory1;
my $path_current1= Cwd::cwd();
print "$path_current1\n";
system ("${repeatmasker_d}trf ${input2}.fasta 2 5 7 80 10 50 2000 -h -f -d -m");
system ("${dust_d}bin/dust ${input2}.fasta.2.5.7.80.10.50.2000.mask >${input2}.fasta2");
if (-e ${input2}.".fasta.2.5.7.80.10.50.2000.mask") {system("rm ${input2}.fasta.2.5.7.80.10.50.2000.mask");}
system ("perl ${directory}Scripts/Exclude_trf_dust_reads061617.pl ${input2}.fasta2 >${input2}.fasta3");
system ("${repeatmasker_d}RepeatMasker -e rmblast -pa $threads -species human -s ${input2}.fasta3");
system ("perl ${directory}Scripts/Reformat_repeatmasker_result.pl ${input2} >${input2}.repeat");
system ("perl ${directory}Scripts/Reformat_trf_dust_result2.pl ${input2} >${input2}.repeat2");
chdir $path_current;
} else {
system ("${repeatmasker_d}trf ${input_sampleID}.fasta 2 5 7 80 10 50 2000 -h -f -d -m");
system ("${dust_d}bin/dust ${input_sampleID}.fasta.2.5.7.80.10.50.2000.mask >${input_sampleID}.fasta2");
if (-e ${input_sampleID}.".fasta.2.5.7.80.10.50.2000.mask") {system ("rm ${input_sampleID}.fasta.2.5.7.80.10.50.2000.mask");}
system ("perl ${directory}Scripts/Exclude_trf_dust_reads061617.pl ${input_sampleID}.fasta2 >${input_sampleID}.fasta3");
system ("${repeatmasker_d}RepeatMasker -e rmblast -pa $threads -species human -s ${input_sampleID}.fasta3");
system ("perl ${directory}Scripts/Reformat_repeatmasker_result.pl ${input_sampleID} >${input_sampleID}.repeat");
system ("perl ${directory}Scripts/Reformat_trf_dust_result2.pl ${input_sampleID} >${input_sampleID}.repeat2");
}
if (-e ${input_sampleID}.".repeat") {system ("rm ${input_sampleID}.repeat");}
if (-e ${input_sampleID}.".fasta3") {system ("rm ${input_sampleID}.fasta3");}
if (-e ${input_sampleID}.".fasta3.cat") {system ("rm ${input_sampleID}.fasta3.cat");}
if (-e ${input_sampleID}.".fasta3.masked") {system ("rm ${input_sampleID}.fasta3.masked");}
if (-e ${input_sampleID}.".fasta3.out") {system ("rm ${input_sampleID}.fasta3.out");}
if (-e ${input_sampleID}.".fasta3.tbl") {system ("rm ${input_sampleID}.fasta3.tbl");}
system ("rm -rf ${input_sampleID}*RMoutput");
if (-e ${input_sampleID}.".fasta.2.5.7.80.10.50.2000.dat") {system ("rm ${input_sampleID}.fasta.2.5.7.80.10.50.2000.dat");}
if (-e ${input_sampleID}.".fasta2") {system ("rm ${input_sampleID}.fasta2");}
}
##### create type file
sub create_type {
system ("${samtools_d}samtools view ${input_sampleID}_sm.bam >${input_sampleID}_sm.sam");
open SM,"${input_sampleID}_sm.sam";
open TYPE,">${input_sampleID}.type";
my $sm_1="";
while($sm_1=<SM>){
my @sm_1=split /\s+/, $sm_1;
if($sm_1[1]%256>=128) {
print TYPE "$sm_1[0] L\n";
} else {
print TYPE "$sm_1[0] R\n";
}
}
close TYPE;
close SM;
}
##### quality control main
sub fastq_qc {
my ($input1,$seq_type1) = @_;
if($seq_type1 eq 1){
fastq_split_qc($input1);
fastq_PE_qc($input1);
} elsif ($seq_type1 eq 2){
fastq_PE_qc($input1);
} elsif ($seq_type1 eq 3){
fastq_split_qc($input1);
if ($mode eq "standard") {
system ("perl ${NGSQCToolkit_d}Trimming/TrimmingReads_sanger.pl -i ${input1}.1fq -q $QS_cutoff -n 30");
system ("perl ${NGSQCToolkit_d}QC/IlluQC_PRLL.pl -se ${input1}.1fq_trimmed 2 1 -c $threads -l 80 -s $QS_cutoff");
my @directory1=split /\//, ${input1};
my $directory1=join("/",@directory1[0..$#directory1-1]);
my $input2=$directory1[$#directory1];
if($#directory1>0){
system ("mv ${directory1}/IlluQC_Filtered_files/${input2}.1fq_trimmed_filtered ${input1}.1fq");
} else {
system ("mv IlluQC_Filtered_files/${input1}.1fq_trimmed_filtered ${input1}.1fq");
}
system ("rm ${input1}.1fq_trimmed");
}
}
}
##### fastq quality control for paired_end_reads
sub fastq_PE_qc {
my ($input1)=@_;
system ("perl ${NGSQCToolkit_d}Trimming/TrimmingReads_sanger.pl -i ${input1}_1.1fq -irev ${input1}_2.1fq -q $QS_cutoff -n 30");
system ("perl ${NGSQCToolkit_d}QC/IlluQC_PRLL.pl -pe ${input1}_1.1fq_trimmed ${input1}_2.1fq_trimmed 2 1 -c $threads -l 80 -s $QS_cutoff");
my @directory1=split /\//, ${input1};
my $directory1=join("/",@directory1[0..$#directory1-1]);
my $input2=$directory1[$#directory1];
if($#directory1>0){
system ("mv ${directory1}/IlluQC_Filtered_files/${input2}_1.1fq_trimmed_filtered ${input1}_1.1fastq");
system ("mv ${directory1}/IlluQC_Filtered_files/${input2}_2.1fq_trimmed_filtered ${input1}_2.1fastq");
} else {
system ("mv IlluQC_Filtered_files/${input1}_1.1fq_trimmed_filtered ${input1}_1.1fastq");
system ("mv IlluQC_Filtered_files/${input1}_2.1fq_trimmed_filtered ${input1}_2.1fastq");
}
system ("echo ${input1}_1.1fastq >${input1}_list");
system ("echo ${input1}_2.1fastq >>${input1}_list");
system ("${fastuniq_d}source/fastuniq -i ${input1}_list -t q -o ${input1}_1.1fq -p ${input1}_2.1fq");
system ("rm ${input1}_list");
system ("rm ${input1}_1.1fq_trimmed");
system ("rm ${input1}_2.1fq_trimmed");
system ("rm ${input1}_1.1fastq");
system ("rm ${input1}_2.1fastq");
}
##### fastq quality control for split reads
sub fastq_split_qc {
my ($input1) = @_;
system ("perl ${NGSQCToolkit_d}Trimming/TrimmingReads_sanger.pl -i ${input1}_1soft.fastq -q $QS_cutoff");
system ("perl ${NGSQCToolkit_d}QC/IlluQC_PRLL.pl -se ${input1}_1soft.fastq_trimmed 2 1 -c $threads -l 80 -s $QS_cutoff");
my @directory1=split /\//, ${input1};
my $directory1=join("/",@directory1[0..$#directory1-1]);
my $input2=$directory1[$#directory1];
if($#directory1>0) {
system ("mv ${directory1}/IlluQC_Filtered_files/${input2}_1soft.fastq_trimmed_filtered ${input1}_1soft.fastq");
} else {
system ("mv IlluQC_Filtered_files/${input1}_1soft.fastq_trimmed_filtered ${input1}_1soft.fastq");
}
system ("rm ${input1}_1soft.fastq_trimmed");
system ("perl ${directory}Scripts/Soft_clipping_filter.pl -length 20 -file ${input_sampleID}_1soft.fastq -o ${input_sampleID}");
system ("rm ${input_sampleID}_1soft.fastq");
}
##### align to the human reference genome
sub align_to_hg {
my ($input1,$suffix1)=@_;
if ($data_type eq "RNA-seq" && $order ne 2){
my $suffix2=$suffix1;
$suffix2=~s/.gz$//;
if($sequencing_type eq "paired-end"){
if($suffix1 =~ "gz"){
system ("gunzip -c ${input1}_1${suffix1} >${input1}_1${suffix2}");
system ("gunzip -c ${input1}_2${suffix1} >${input1}_2${suffix2}");
}
system ("${tophat_d}tophat -p $threads -o ${input1} $human_genome_tophat ${input1}_1${suffix2} ${input1}_2${suffix2}");
} else {
if($suffix1 =~ "gz"){