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fast-plast.pl
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#!/usr/bin/env perl
use strict;
use warnings;
use Getopt::Long;
use Pod::Usage;
use FindBin;
use lib ("$FindBin::Bin/PerlLib");
use File::Spec;
use Cwd;
use Cwd 'abs_path';
use Env qw (PATH);
use IO::Uncompress::Gunzip qw(gunzip $GunzipError) ;
BEGIN {
$ENV{FP_HOME} = "$FindBin::Bin";
}
###directories
my $FPROOT = "$FindBin::RealBin";
my $AFIN_DIR = "$FPROOT/afin";
my $COVERAGE_DIR = "$FPROOT/Coverage_Analysis";
my $FPBIN = "$FPROOT/bin";
my $TRIMMOMATIC; #path to trimmomatic executable
my $BOWTIE2; #path to bowtie2 executable
my $SPADES; #path to spades executable
my $BLAST; #path to blast executable
my $SSPACE; #path to sspace exectuable
my $BOWTIE1; #path to bowtie1 executable
my $JELLYFISH; #path to jellyfish2 excecutable
$ENV{'PATH'} = $PATH.':'.$BOWTIE1;
my $help;
my $paired_end1;
my $paired_end2;
my $single_end;
my $name="Fast-Plast";
my $bowtie_index = "All";
my $posgenes= $FPBIN . "/Angiosperm_Chloroplast_Genes.fsa";
my $coverage_check;
my $min_coverage;
my $threads = 4;
my $adapters = $FPBIN . "/adapters/NEB-PE.fa";
my $version;
my $current_version = "Fast-Plast v.1.2.7";
my $user_bowtie;
my $clean;
my $subsample;
my $cov_only;
my $skip;
GetOptions('help|?' => \$help,'version' => \$version, "1=s" => \$paired_end1, "2=s" => \$paired_end2, "single=s" => \$single_end, "bowtie_index=s" => \$bowtie_index, "user_bowtie=s" => \$user_bowtie, "name=s" => \$name, "clean=s" => \$clean, 'coverage_analysis' => \$coverage_check, 'skip=s' => \$skip, 'positional_genes' => \$posgenes, "threads=i" => \$threads, "min_coverage=i" => \$min_coverage, "adapters=s" => \$adapters, "subsample=i" => \$subsample, "only_coverage=s" => \$cov_only) or pod2usage( { -message => "ERROR: Invalid parameter." } );
if($version) {
pod2usage( { -verbose => 99, -sections => "VERSION" } );
}
if ($help) {
pod2usage( { -exitstatus => 0 } );
}
if ( !$paired_end1 && !$single_end ) {
pod2usage( { -message => "ERROR: Missing reads file(s)." } );
}
if (!$paired_end1 && $paired_end2 || !$paired_end2 && $paired_end1){
pod2usage( { -message => "ERROR: Missing other paired end file." } );
}
if ( !$name ) {
pod2usage( { -message => "ERROR: Missing sample name." } );
}
if($user_bowtie){
if ( !glob($user_bowtie."*")) {
pod2usage( { -message => "ERROR: User supplied Bowtie2 indices do not exist. Check path." } );
}
}
### Get full paths for files. Glob would work for all of them, but it requires perl 5.6+. Only using it for the ~ calls, just in case. ####
my $datestring = localtime();
my $start_time = time;
open(STDERR, '>', $name.'_results_error.log') or die "Can't open log.\n";
open(STDOUT, '>', $name.'_results_out.log') or die "Can't open log.\n";
open my $LOGFILE, ">", $name."_Fast-Plast_Progress.log" or die "Can't open log.\n";
print $LOGFILE "$datestring\tStarting $current_version.\n";
my @p1_array;
if($paired_end1){
my @temp_array = split(",", $paired_end1);
for my $tfile (@temp_array){
if($tfile =~ /~/){
$tfile = glob ($tfile);
}
my $abs_path = abs_path($tfile);
unless(-e $tfile){
die "$tfile does not exist.";
}
unless(-r $tfile){
die "$tfile is not readable.";
}
push(@p1_array, $abs_path);
}
}
my @p2_array;
if($paired_end2){
my @temp_array = split(",", $paired_end2);
for my $tfile (@temp_array){
if($tfile =~ /~/){
$tfile = glob ($tfile);
}
unless(-e $tfile){
die "$tfile does not exist.";
}
unless(-r $tfile){
die "$tfile is not readable.";
}
my $abs_path = abs_path($tfile);
push(@p2_array, $abs_path);
}
}
my @s_array;
if($single_end){
my @temp_array = split(",", $single_end);
for my $tfile (@temp_array){
if($tfile =~ /~/){
$tfile = glob ($tfile);
}
unless(-e $tfile){
die "$tfile does not exist.";
}
unless(-r $tfile){
die "$tfile is not readable.";
}
my $abs_path = abs_path($tfile);
push(@s_array, $abs_path);
}
}
my $pe_libs = @p1_array;
my $s_libs = @s_array;
if(!$pe_libs){
$pe_libs = 0;
}
if(!$s_libs){
$s_libs = 0;
}
print $LOGFILE "\t\t\t\tAssemblying plastome with $s_libs single end libraries and $pe_libs paired end libraries.\n";
###Get read size###
my $current_runtime = localtime();
print $LOGFILE "$current_runtime\tDetermining best kmer sizes.\n";
my $maxsize=0;
if(@p1_array){
for my $file (@p1_array){
open my $tfile, "<", $file;
my $count = 0;
SIZE: while(<$tfile>){
my $seq = readline($tfile);
chomp($seq);
if(length($seq) > $maxsize){
$maxsize = length($seq);
}
$count++;
if($count == 100){
last SIZE;
}
readline($tfile);
readline($tfile);
}
}
}
if(@p2_array){
for my $file (@p2_array){
open my $tfile, "<", $file;
my $count = 0;
SIZE: while(<$tfile>){
my $seq = readline($tfile);
chomp($seq);
if(length($seq) > $maxsize){
$maxsize = length($seq);
}
$count++;
if($count == 100){
last SIZE;
}
readline($tfile);
readline($tfile);
}
}
}
if(@s_array){
for my $file (@s_array){
open my $tfile, "<", $file;
my $count = 0;
SIZE: while(<$tfile>){
my $seq = readline($tfile);
chomp($seq);
if(length($seq) > $maxsize){
$maxsize = length($seq);
}
$count++;
if($count == 100){
last SIZE;
}
readline($tfile);
readline($tfile);
}
}
}
##########
###Set K-mer Size###
my $spades_kmer;
if($maxsize >= 140){
$spades_kmer = "55,87,121";
}
elsif($maxsize >= 100){
$spades_kmer = "55,69,87";
}
elsif($maxsize >= 80){
$spades_kmer = "45,57,69";
}
elsif($maxsize >= 50){
$spades_kmer = "31,37,43";
}
else{
$spades_kmer = "23,27,31";
}
print $LOGFILE "\t\t\t\tK-mer sizes for SPAdes set at $spades_kmer.\n";
##########
########## Create Directory ###########
mkdir("$name");
chdir("$name");
open my $SUMMARY, ">", $name."_Plastome_Summary.txt";
print $SUMMARY "Sample:\t$name\nFast-Plast Version:\t$current_version\n";
########## Start Trimmomatic ##########
$current_runtime = localtime();
print $LOGFILE "$current_runtime\tStarting read trimming with Trimmomatic.\n\t\t\t\tUsing $TRIMMOMATIC.\n";
mkdir("1_Trimmed_Reads");
chdir("1_Trimmed_Reads");
###Subsample Data###
my $total_input_files;
if(@p1_array){
$total_input_files+=scalar @p1_array;
}
if(@p2_array){
$total_input_files+=scalar @p2_array;
}
if(@s_array){
$total_input_files+=scalar @s_array;
}
if($subsample){
my $readsperfile = $subsample/$total_input_files;
$readsperfile = int($readsperfile);
if(@p1_array){
open my $sub_p1, ">", "subset_file1.fq";
for my $p1f (@p1_array){
my $count = 0;
my $tfile;
if($p1f =~ /\.gz/){
$tfile = IO::Uncompress::Gunzip->new($p1f) or die "Problem found with $p1f: $GunzipError";
}
else{
open $tfile, "<", $p1f;
}
SUB: while(<$tfile>){
chomp;
if($count <= $readsperfile*4){
print $sub_p1 "$_\n";
$count++;
}
else{
last SUB;
}
}
}
}
if(@p2_array){
open my $sub_p2, ">", "subset_file2.fq";
for my $p2f (@p2_array){
my $count = 0;
my $tfile;
if($p2f =~ /\.gz/){
$tfile = IO::Uncompress::Gunzip->new($p2f) or die "Problem found with $p2f: $GunzipError";
}
else{
open $tfile, "<", $p2f;
}
SUB: while(<$tfile>){
chomp;
if($count <= $readsperfile*4){
print $sub_p2 "$_\n";
$count++;
}
else{
last SUB;
}
}
}
}
if(@s_array){
open my $sub_s, ">", "subset_files.fq";
for my $psf (@s_array){
my $count = 0;
my $tfile;
if($psf =~ /\.gz/){
$tfile = IO::Uncompress::Gunzip->new($psf, MultiStream => 1) or die "Problem found with $psf: $GunzipError";
}
else{
open $tfile, "<", $psf;
}
SUB: while(<$tfile>){
chomp;
if($count <= $readsperfile*4){
print $sub_s "$_\n";
$count++;
}
else{
last SUB;
}
}
}
}
@p1_array=();
push(@p1_array, "subset_file1.fq");
@p2_array=();
push(@p2_array, "subset_file2.fq");
@s_array=();
push(@s_array, "subset_files.fq");
}
if($skip && $skip eq "trim"){
if(@p1_array){
my $tfile;
for my $p1array (@p1_array){
if($p1array =~ /\.gz/){
$tfile = IO::Uncompress::Gunzip->new($p1array, MultiStream => 1) or die "Problem found with $p1array: $GunzipError";
}
else{
$tfile = $p1array;
}
`cat $tfile >> $name.trimmed_P1.fq`;
}
}
if(@p2_array){
my $tfile;
for my $p2array (@p2_array){
if($p2array =~ /\.gz/){
$tfile = IO::Uncompress::Gunzip->new($p2array, MultiStream => 1) or die "Problem found with $p2array: $GunzipError";
}
else{
$tfile = $p2array;
}
`cat $tfile >> $name.trimmed_P2.fq`;
}
}
if(@s_array){
my $tfile;
for my $sarray (@s_array){
if($sarray =~ /\.gz/){
$tfile = IO::Uncompress::Gunzip->new($sarray, MultiStream => 1) or die "Problem found with $sarray: $GunzipError";
}
else{
$tfile = $sarray;
}
`cat $tfile >> $name.trimmed_UP.fq`;
}
}
if (-s $name.".trimmed_P1.fq"){
my $se_size = &count_lines($name.".trimmed_P1.fq");
chomp($se_size);
$se_size=($se_size/4)*2;
print $SUMMARY "Total Cleaned Pair-End Reads:\t$se_size\n";
}
if (-s $name.".trimmed_UP.fq"){
my $se_size = &count_lines($name.".trimmed_UP.fq");
chomp($se_size);
$se_size=$se_size/4;
print $SUMMARY "Total Cleaned Single End Reads:\t$se_size\n";
}
}
else{
if($adapters =~ /nextera/i){
$adapters=$FPBIN."/adapters/NexteraPE-PE.fa";
}
if($adapters =~ /truseq/i){
$adapters=$FPBIN."/adapters/TruSeq3-PE.fa";
}
if($adapters =~ /NEB/i){
$adapters=$FPBIN."/adapters/NEB-PE.fa";
}
if(@p1_array){
for (my $i=0; $i < $pe_libs; $i++){
my $trim_exec = "java -classpath " . $TRIMMOMATIC . " org.usadellab.trimmomatic.TrimmomaticPE -threads " . $threads . " " . $p1_array[$i] . " " . $p2_array[$i] . " " . $name."_".$i.".trimmed_P1.fq " . $name."_".$i.".trimmed_U1.fq " . $name."_".$i.".trimmed_P2.fq " . $name."_".$i.".trimmed_U2.fq " . "ILLUMINACLIP:".$adapters.":1:30:10 SLIDINGWINDOW:10:20 MINLEN:40";
system($trim_exec);
}
`cat $name\*trimmed_P1.fq > $name.trimmed_P1.fq`;
`cat $name\*trimmed_P2.fq > $name.trimmed_P2.fq`;
if(glob("$name*trimmed_U*.fq")){
`cat $name\*trimmed_U*.fq > $name.trimmed_UP.fq`;
}
}
if(@s_array){
for (my $i=0; $i < $s_libs; $i++){
my $trim_exec = "java -classpath " . $TRIMMOMATIC . " org.usadellab.trimmomatic.TrimmomaticSE -threads " . $threads . " " . $s_array[$i] . " " . $name."_".$i.".trimmed_SE.fq " . "ILLUMINACLIP:".$adapters.":1:30:10 SLIDINGWINDOW:10:20 MINLEN:40";
system($trim_exec);
}
`cat $name\*trimmed_SE.fq >> $name.trimmed_UP.fq`;
}
if (-s $name.".trimmed_P1.fq"){
my $se_size = &count_lines($name.".trimmed_P1.fq");
chomp($se_size);
$se_size=($se_size/4)*2;
print $SUMMARY "Total Cleaned Pair-End Reads:\t$se_size\n";
}
if (-s $name.".trimmed_UP.fq"){
my $se_size = &count_lines($name.".trimmed_UP.fq");
chomp($se_size);
$se_size=$se_size/4;
print $SUMMARY "Total Cleaned Single End Reads:\t$se_size\n";
}
unlink(glob("$name\_*"));
my @tfile_read = glob("$name.trimmed*");
for my $check_tfile (@tfile_read){
if(-z $check_tfile){
unlink($check_tfile);
}
}
}
chdir("../");
##########
if($cov_only){
$current_runtime = localtime();
print $LOGFILE "$current_runtime\tStarting coverage analyses.\n";
my $check_finish = $cov_only;
unless(-e $check_finish){
print $LOGFILE "\t\t\t\tCannot complete coverage analysis. File empty or not found.";
die;
}
mkdir("Coverage_Analysis");
chdir("Coverage_Analysis");
my $build_bowtie2_exec = $BOWTIE2 . "-build $cov_only " . $name . "_bowtie";
system($build_bowtie2_exec);
my $cov_bowtie2_exec;
if(glob("../1_Trimmed_Reads/$name*trimmed_U*.fq") && glob("../1_Trimmed_Reads/$name*trimmed_P*.fq")){
$cov_bowtie2_exec = $BOWTIE2 . " --very-sensitive-local --quiet --al map_hits.fq --al-conc map_pair_hits.fq -p " . $threads . " -x " . $name ."_bowtie" . " -1 ../1_Trimmed_Reads/" . $name . ".trimmed_P1.fq -2 ../1_Trimmed_Reads/" . $name . ".trimmed_P2.fq -U ../1_Trimmed_Reads/" . $name . ".trimmed_UP.fq -S " . $name . ".sam";
}
elsif(glob("../1_Trimmed_Reads/$name*trimmed_U*.fq") && !glob("../1_Trimmed_Reads/$name*trimmed_P*.fq")){
$cov_bowtie2_exec = $BOWTIE2 . " --very-sensitive-local --quiet --al map_hits.fq -p " . $threads . " -x " . $name ."_bowtie" . " -U ../1_Trimmed_Reads/" . $name . ".trimmed_UP.fq -S " . $name . ".sam";
}
elsif(!glob("../1_Trimmed_Reads/$name*trimmed_U*.fq") && glob("../1_Trimmed_Reads/$name*trimmed_P*.fq")){
$cov_bowtie2_exec = $BOWTIE2 . " --very-sensitive-local --quiet --al map_hits.fq --al-conc map_pair_hits.fq -p " . $threads . " -x " . $name ."_bowtie" . " -1 ../1_Trimmed_Reads/" . $name . ".trimmed_P1.fq -2 ../1_Trimmed_Reads/" . $name . ".trimmed_P2.fq -S " . $name . ".sam";
}
else{
print $LOGFILE "Could not find reads to complete Coverage Analysis. Check 1_Trimmed_Reads.\n";
die "Could not find reads to complete Coverage Analysis. Check 1_Trimmed_Reads.\n";
}
system($cov_bowtie2_exec);
my $jellyfish_count_exec = $JELLYFISH . " count -m 25 -t ". $threads . " -C -s 1G map_*";
system($jellyfish_count_exec);
my $jellyfish_dump_exec = $JELLYFISH . " dump mer_counts.jf > " . $name . "_25dump";
system($jellyfish_dump_exec);
my $window_cov_exec = "perl " . $COVERAGE_DIR . "/new_window_coverage.pl " . $name . "_25dump $cov_only 25";
system($window_cov_exec);
my $rscript_exec = "Rscript " . $COVERAGE_DIR . "/plot_coverage.r " . $name . ".coverage_25kmer.txt ". $name;
system($rscript_exec);
my $check_cov_exec;
if(defined $min_coverage){
$check_cov_exec = "perl " . $COVERAGE_DIR . "/check_plastid_coverage.pl " . $name . ".coverage_25kmer.txt 25 ".$min_coverage;
}
else{
$check_cov_exec = "perl " . $COVERAGE_DIR . "/check_plastid_coverage.pl " . $name . ".coverage_25kmer.txt 25";
}
my $coverage_used = `$check_cov_exec`;
chomp($coverage_used);
$current_runtime = localtime();
print $LOGFILE "$current_runtime\tMinimum coverage of $coverage_used for verifying assembly.\n";
print $SUMMARY "Minimum Coverage Used for Verification: $coverage_used\n";
chdir("../");
$current_runtime = localtime();
print $LOGFILE "$current_runtime\tCoverage analysis finished.\n";
if(-z "Coverage_Analysis/".$name."_problem_regions_plastid_assembly.txt"){
print $LOGFILE "\t\t\t\tNo issues with assembly coverage were identified.\n";
coverage_summary($cov_only, "Coverage_Analysis/");
if($clean){
if ($clean eq "light"){
unlink(glob("5_Plastome_Finishing/*fsa.n*"));
unlink(glob("*/*bt2"));
unlink(glob("Coverage_Analysis/*25dump"));
unlink(glob("Coverage_Analysis/mer_counts.jf"));
unlink(glob("*/*.sam"));
}
if ($clean eq "deep"){
rmdir("1_Trimmed_Reads");
rmdir("2_Bowtie_Mapping");
rmdir("3_Spades_Assembly");
rmdir("4_Afin_Assembly");
rmdir("5_Plastome_Finishing");
unlink(glob("Coverage_Analysis/*25dump"));
unlink(glob("Coverage_Analysis/mer_counts.jf"));
unlink(glob("*/*bt2"));
}
}
die "Fast-Plast finished.\n";
}
else{
print $LOGFILE "\t\t\t\tProblem regions identified with coverage analysis. Check output.\n";
die "Fast-Plast finished.\n";
}
}
########## Start Bowtie2 ##########
$current_runtime = localtime();
print $LOGFILE "$current_runtime\tStarting read mapping with bowtie2.\n\t\t\t\tUsing $BOWTIE2.\n";
mkdir("2_Bowtie_Mapping");
chdir("2_Bowtie_Mapping");
if($user_bowtie){
$bowtie_index = $user_bowtie;
}
else{
$bowtie_index= &build_bowtie2_indices($bowtie_index);
}
my $bowtie2_exec;
if(glob("../1_Trimmed_Reads/$name*trimmed_U*.fq") && glob("../1_Trimmed_Reads/$name*trimmed_P*.fq")){
$bowtie2_exec = $BOWTIE2 . " --very-sensitive-local --al map_hits.fq --al-conc map_pair_hits.fq -p " . $threads . " -x " . $bowtie_index . " -1 ../1_Trimmed_Reads/" . $name . ".trimmed_P1.fq -2 ../1_Trimmed_Reads/" . $name . ".trimmed_P2.fq -U ../1_Trimmed_Reads/" . $name . ".trimmed_UP.fq -S " . $name . ".sam";
}
elsif(!glob("../1_Trimmed_Reads/$name*trimmed_U*.fq") && glob("../1_Trimmed_Reads/$name*trimmed_P*.fq")){
$bowtie2_exec = $BOWTIE2 . " --very-sensitive-local --al map_hits.fq --al-conc map_pair_hits.fq -p " . $threads . " -x " . $bowtie_index . " -1 ../1_Trimmed_Reads/" . $name . ".trimmed_P1.fq -2 ../1_Trimmed_Reads/" . $name . ".trimmed_P2.fq -S " . $name . ".sam";
}
elsif(glob("../1_Trimmed_Reads/$name*trimmed_U*.fq")){
$bowtie2_exec = $BOWTIE2 . " --very-sensitive-local --al map_hits.fq -p " . $threads . " -x " . $bowtie_index . " -U ../1_Trimmed_Reads/" . $name . ".trimmed_UP.fq -S " . $name . ".sam";
}
else{
print $LOGFILE "\t\t******************ERROR: No trimmed read files were identified to run SPAdes. Please check 1_Trimmed_Reads.******************\n";
die ("No trimmed read files were identified to run SPAdes. Please check 1_Trimmed_Reads.\n");
}
system($bowtie2_exec);
if (-s "map_pair_hits.1.fq"){
my $se_size = &count_lines("map_pair_hits.1.fq");
chomp($se_size);
$se_size=($se_size/4)*2;
print $SUMMARY "Total Concordantly Mapped Reads:\t$se_size\n";
}
if (-s "map_hits.fq"){
my $se_size = &count_lines("map_hits.fq");
chomp($se_size);
$se_size=$se_size/4;
print $SUMMARY "Total Non-concordantly Mapped Reads:\t$se_size\n";
}
chdir("../");
########## Start SPAdes ##########
$current_runtime = localtime();
print $LOGFILE "$current_runtime\tStarting initial assembly with SPAdes.\n\t\t\t\tUsing $SPADES.\n";
mkdir("3_Spades_Assembly");
chdir("3_Spades_Assembly");
my $spades_exec;
if(-s "../2_Bowtie_Mapping/map_pair_hits.1.fq" && -s "../2_Bowtie_Mapping/map_pair_hits.2.fq" && -s "../2_Bowtie_Mapping/map_hits.fq"){
$spades_exec = "python " . $SPADES . " -o spades_iter1 -1 ../2_Bowtie_Mapping/map_pair_hits.1.fq -2 ../2_Bowtie_Mapping/map_pair_hits.2.fq -s ../2_Bowtie_Mapping/map_hits.fq --only-assembler -k " . $spades_kmer . " -t " . $threads;
}
elsif(-s "../2_Bowtie_Mapping/map_pair_hits.1.fq" && -s "../2_Bowtie_Mapping/map_pair_hits.2.fq" && -z ("../2_Bowtie_Mapping/map_hits.fq" || ! -e "../2_Bowtie_Mapping/map_hits.fq")){
$spades_exec = "python " . $SPADES . " -o spades_iter1 -1 ../2_Bowtie_Mapping/map_pair_hits.1.fq -2 ../2_Bowtie_Mapping/map_pair_hits.2.fq --only-assembler -k " . $spades_kmer . " -t " . $threads;
}
elsif (-s "../2_Bowtie_Mapping/map_hits.fq"){
$spades_exec = "python " . $SPADES . " -o spades_iter1 -s ../2_Bowtie_Mapping/map_hits.fq --only-assembler -k " . $spades_kmer . " -t " . $threads;
}
else{
print $LOGFILE "\t\t******************ERROR: No mapped reads files were identified to run SPAdes. Please check 2_Bowtie_Mapping.******************\n";
die ("No mapped reads files were identified to run SPAdes. Please check 2_Bowtie_Mapping.\n");
}
system($spades_exec);
chdir("../");
########## Start Afin ##########
$current_runtime = localtime();
print $LOGFILE "$current_runtime\tStarting improved assembly with afin.\n";
mkdir("4_Afin_Assembly");
chdir("4_Afin_Assembly");
`perl $FPBIN/filter_spades_contigs_weigthed.pl ../3_Spades_Assembly/spades_iter1/contigs.fasta`;
my %temp_filtered;
my $temp_filter_id;
open my $file_filter, "<", "filtered_spades_contigs.fsa";
open my $out_filter, ">", "temp_filtered_spades_contigs.fsa";
while(<$file_filter>){
chomp;
if(/>/){
$temp_filter_id=$_;
}
else{
$temp_filtered{$temp_filter_id}.=$_;
}
}
my @bases = ("A", "C", "T", "G");
my %repeats;
for my $base (@bases){
for my $base2 (@bases){
$repeats{$base.$base2}=0;
$repeats{$base2.$base}=0;
}
}
for my $temp_seq (keys %temp_filtered){
my %temprepeats=%repeats;
for my $re (keys %repeats){
for (my $i =0; $i <= length($temp_filtered{$temp_seq})-1; $i++){
my $result = index($temp_filtered{$temp_seq}, $re, $i);
if($result >= 0){
$temprepeats{$re}++;
$i = $result;
}
}
}
my $max_repeat=0;
for my $re_val (keys %temprepeats){
if($temprepeats{$re_val} >$max_repeat){
$max_repeat = $temprepeats{$re_val};
}
}
if($max_repeat/(length($temp_filtered{$temp_seq})/2) > 0.5){
next;
}
else{
print $out_filter "$temp_seq\n$temp_filtered{$temp_seq}\n";
}
}
rename("temp_filtered_spades_contigs.fsa", "filtered_spades_contigs.fsa");
my $current_afin;
my $extension = $maxsize*0.75;
my ($total_afin_contigs, $max_afin, $min_afin) = &run_afin("150,50,50",100,"20,15,10","2,1,1","filtered_spades_contigs.fsa",$extension);
print $LOGFILE "\t\t\t\tAfter afin, there are $total_afin_contigs contigs with a maximum size of $max_afin and a minimum size of $min_afin.\n";
$current_runtime = localtime();
print $LOGFILE "$current_runtime\tRemoving nested contigs.\n";
my $gotofinish;
if( $total_afin_contigs > 1){
$current_afin = $name . "_afin_iter2.fa";
`$BLAST/makeblastdb -in $current_afin -dbtype nucl`;
my $blast_afin_exec = $BLAST . "blastn -query " . $current_afin . " -db " . $current_afin . " -evalue 1e-40 -outfmt 6 -max_target_seqs 100000000 > " . $current_afin . ".blastn";
`$blast_afin_exec`;
&remove_nested($current_afin, $current_afin.".blastn");
$total_afin_contigs = &count_contigs($current_afin);
my ($percent_recovered_genes, $contigs_db_genes) = &cpgene_recovery($current_afin);
my %contigs_db_genes = %$contigs_db_genes;
if ($total_afin_contigs > 1){
&remove_contamination($current_afin, \%contigs_db_genes);
}
$total_afin_contigs = &count_contigs($current_afin);
($percent_recovered_genes, $contigs_db_genes) = &cpgene_recovery($current_afin);
%contigs_db_genes = %$contigs_db_genes;
if ($total_afin_contigs > 1){
($percent_recovered_genes, $contigs_db_genes) = &cpgene_recovery($current_afin);
%contigs_db_genes = %$contigs_db_genes;
$percent_recovered_genes=$percent_recovered_genes*100;
print $LOGFILE "\t\t\t\tChecking coverage of afin output with $total_afin_contigs contigs after contamination removal.\n";
print $LOGFILE "\t\t\t\t$percent_recovered_genes\% of known angiosperm chloroplast genes were recovered in $current_afin.\n";
if(@p1_array){
$current_afin = &scaffolding($current_afin,$name);
rename($current_afin, $name.".final.scaffolds.fasta");
$current_afin=$name.".final.scaffolds.fasta";
$current_afin=&afin_wrap($current_afin, "extend");
`$BLAST/makeblastdb -in $current_afin -dbtype nucl`;
$blast_afin_exec = $BLAST . "blastn -query " . $current_afin . " -db " . $current_afin . " -evalue 1e-40 -outfmt 6 -max_target_seqs 100000000 > " . $current_afin . ".blastn";
`$blast_afin_exec`;
&remove_nested($current_afin, $current_afin.".blastn");
($percent_recovered_genes, $contigs_db_genes) = &cpgene_recovery($current_afin);
%contigs_db_genes = %$contigs_db_genes;
&remove_contamination($current_afin, \%contigs_db_genes);
print $LOGFILE "\t\t\t\tRemoved mitochondrial-like sequences.\n";
$total_afin_contigs = &count_contigs($current_afin);
if($total_afin_contigs > 1){
open my $cpcomposition, ">", "Chloroplast_gene_composition_of_final_contigs.txt";
for my $contig_name (sort keys %contigs_db_genes){
for my $gene_name (sort keys %{$contigs_db_genes{$contig_name}}){
print $cpcomposition "$contig_name\t$gene_name\n";
}
}
close ($cpcomposition);
mkdir("../Final_Assembly");
rename($current_afin, "../Final_Assembly/".$current_afin);
rename("Chloroplast_gene_composition_of_final_contigs.txt", "../Final_Assembly/Chloroplast_gene_composition_of_final_contigs.txt");
chdir("../Final_Assembly");
my $temppwd = `pwd`;
chomp($temppwd);
$temppwd .= "/". $current_afin;
print $LOGFILE "\t\t\t\tCannot scaffold contigs into a single piece. Coverage is too low or poorly distributed across plastome. Best contigs are in $temppwd\. A list of genes in each contig can be found in \"Chloroplast_gene_composition_of_final_contigs.txt\"\.\n";
die;
}
else{
my ($percent_recovered_genes, $contigs_db_genes) = &cpgene_recovery($current_afin);
my %contigs_db_genes = %$contigs_db_genes;
$percent_recovered_genes=$percent_recovered_genes*100;
print $LOGFILE "\t\t\t\tChecking coverage of scaffolded contigs with $total_afin_contigs.\n";
print $LOGFILE "\t\t\t\t$percent_recovered_genes\% of known angiosperm chloroplast genes were recovered in $current_afin.\n";
open my $cpcomposition, ">", "Chloroplast_gene_composition_of_scaffolded_contigs.txt";
for my $contig_name (sort keys %contigs_db_genes){
for my $gene_name (sort keys %{$contigs_db_genes{$contig_name}}){
print $cpcomposition "$contig_name\t$gene_name\n";
}
}
close ($cpcomposition);
}
}
else{
open my $cpcomposition, ">", "Chloroplast_gene_composition_of_final_contigs.txt";
for my $contig_name (sort keys %contigs_db_genes){
for my $gene_name (sort keys %{$contigs_db_genes{$contig_name}}){
print $cpcomposition "$contig_name\t$gene_name\n";
}
}
close ($cpcomposition);
mkdir("../Final_Assembly");
rename($current_afin, "../Final_Assembly/".$current_afin);
rename("Chloroplast_gene_composition_of_final_contigs.txt", "../Final_Assembly/Chloroplast_gene_composition_of_final_contigs.txt");
chdir("../Final_Assembly");
my $temppwd = `pwd`;
chomp($temppwd);
$temppwd .= "/". $current_afin;
print $LOGFILE "\t\t\t\tCannot scaffold contigs into a single piece. Coverage is too low or poorly distributed across plastome. Best contigs are in $temppwd\. A list of genes in each contig can be found in \"Chloroplast_gene_composition_of_final_contigs.txt\"\.\n";
die;
}
}
else{
my ($percent_recovered_genes, $contigs_db_genes) = &cpgene_recovery($current_afin);
my %contigs_db_genes = %$contigs_db_genes;
$percent_recovered_genes=$percent_recovered_genes*100;
print $LOGFILE "\t\t\t\tChecking coverage of afin output with $total_afin_contigs contigs.\n";
print $LOGFILE "\t\t\t\t$percent_recovered_genes\% of known angiosperm chloroplast genes were recovered in $current_afin.\n";
open my $cpcomposition, ">", "Chloroplast_gene_composition_of_afin_contigs_nested_removed.txt";
for my $contig_name (sort keys %contigs_db_genes){
for my $gene_name (sort keys %{$contigs_db_genes{$contig_name}}){
print $cpcomposition "$contig_name\t$gene_name\n";
}
}
close ($cpcomposition);
}
}
else{
$current_afin = $name . "_afin_iter2.fa";
my ($percent_recovered_genes, $contigs_db_genes) = &cpgene_recovery($current_afin);
my %contigs_db_genes = %$contigs_db_genes;
$percent_recovered_genes=$percent_recovered_genes*100;
print $LOGFILE "\t\t\t\tChecking coverage of afin output with $total_afin_contigs contigs after contamination removal.\n";
print $LOGFILE "\t\t\t\t$percent_recovered_genes\% of known angiosperm chloroplast genes were recovered in $current_afin.\n";
open my $cpcomposition, ">", "Chloroplast_gene_composition_of_afin_contigs.txt";
for my $contig_name (sort keys %contigs_db_genes){
for my $gene_name (sort keys %{$contigs_db_genes{$contig_name}}){
print $cpcomposition "$contig_name\t$gene_name\n";
}
}
close ($cpcomposition);
}
chdir("../");
########## Start Plastome Finishing ##########
$current_runtime = localtime();
print $LOGFILE "$current_runtime\tStarting plastome finishing.\n\t\t\t\tUsing $posgenes for LSC, SSC, and IR identification.\n";
mkdir("5_Plastome_Finishing");
chdir("5_Plastome_Finishing");
&orientate_plastome("../4_Afin_Assembly/".$current_afin, $name, "../Final_Assembly/");
chdir("../");
if(!-d "Final_Assembly"){
mkdir("Final_Assembly");
rename("4_Afin_Assembly/".$current_afin, "Final_Assembly/".$current_afin);
my ($percent_recovered_genes, $contigs_db_genes) = &cpgene_recovery("Final_Assembly/".$current_afin);
my %contigs_db_genes = %$contigs_db_genes;
$percent_recovered_genes=$percent_recovered_genes*100;
print $LOGFILE "\t\t\t\tChecking coverage of final assembly. Final assembly is the last afin iteration.\n";
print $LOGFILE "\t\t\t\t$percent_recovered_genes\% of known angiosperm chloroplast genes were recovered in $current_afin.\n";
open my $cpcomposition, ">", "Final_Assembly/Chloroplast_gene_composition_of_afin_contigs.txt";
for my $contig_name (sort keys %contigs_db_genes){
for my $gene_name (sort keys %{$contigs_db_genes{$contig_name}}){
print $cpcomposition "$contig_name\t$gene_name\n";
}
}
close ($cpcomposition);
if( -e "4_Afin_Assembly/Chloroplast_gene_composition_of_afin_contigs.txt"){
my $temppwd = `pwd`;
chomp($temppwd);
$temppwd .= "/Final_Assembly/". $current_afin;
print $LOGFILE "\t\t\t\tCould not properly orientate the plastome. Either your plastome does not have an IR or there was an issue with the assembly. Best contigs are in $temppwd\. A list of genes in each contig can be found in \"Chloroplast_gene_composition_of_final_contigs.txt\"\.\n";
die;
}
if( -e "4_Afin_Assembly/Chloroplast_gene_composition_of_afin_contigs_nested_removed.txt"){
my $temppwd = `pwd`;
chomp($temppwd);
$temppwd .= "/Final_Assembly/". $current_afin;
print $LOGFILE "\t\t\t\tCould not properly orientate the plastome. Either your plastome does not have an IR or there was an issue with the assembly. Best contigs are in $temppwd\. A list of genes in each contig can be found in \"Chloroplast_gene_composition_of_final_contigs.txt\"\.\n";
die;
}
}
$current_runtime = localtime();
print $LOGFILE "$current_runtime\tAssembly finished. Check Final_Assembly directory for chloroplast assembly and accessory files.\n";
########## Start Coverage Analysis ##########
if($coverage_check){
$current_runtime = localtime();
print $LOGFILE "$current_runtime\tStarting coverage analyses.\n";
my $check_finish = "Final_Assembly/".$name."_FULLCP.fsa";
unless(-e $check_finish){
print $LOGFILE "\t\t\t\tCannot complete coverage analysis. Full chloroplast genome not complete.";
die;
}
mkdir("Coverage_Analysis");
chdir("Coverage_Analysis");
my $build_bowtie2_exec = $BOWTIE2 . "-build ../Final_Assembly/" . $name . "_FULLCP.fsa " . $name . "_bowtie";
system($build_bowtie2_exec);
my $cov_bowtie2_exec;
if(glob("../1_Trimmed_Reads/$name*trimmed_U*.fq") && glob("../1_Trimmed_Reads/$name*trimmed_P*.fq")){
$cov_bowtie2_exec = $BOWTIE2 . " --very-sensitive-local --quiet --al map_hits.fq --al-conc map_pair_hits.fq -p " . $threads . " -x " . $name ."_bowtie" . " -1 ../1_Trimmed_Reads/" . $name . ".trimmed_P1.fq -2 ../1_Trimmed_Reads/" . $name . ".trimmed_P2.fq -U ../1_Trimmed_Reads/" . $name . ".trimmed_UP.fq -S " . $name . ".sam";
}
elsif(glob("../1_Trimmed_Reads/$name*trimmed_U*.fq") && !glob("../1_Trimmed_Reads/$name*trimmed_P*.fq")){
$cov_bowtie2_exec = $BOWTIE2 . " --very-sensitive-local --quiet --al map_hits.fq -p " . $threads . " -x " . $name ."_bowtie" . " -U ../1_Trimmed_Reads/" . $name . ".trimmed_UP.fq -S " . $name . ".sam";
}
elsif(!glob("../1_Trimmed_Reads/$name*trimmed_U*.fq") && glob("../1_Trimmed_Reads/$name*trimmed_P*.fq")){
$cov_bowtie2_exec = $BOWTIE2 . " --very-sensitive-local --quiet --al map_hits.fq --al-conc map_pair_hits.fq -p " . $threads . " -x " . $name ."_bowtie" . " -1 ../1_Trimmed_Reads/" . $name . ".trimmed_P1.fq -2 ../1_Trimmed_Reads/" . $name . ".trimmed_P2.fq -S " . $name . ".sam";
}
else{
print $LOGFILE "Could not find reads to complete Coverage Analysis. Check 1_Trimmed_Reads.\n";
die "Could not find reads to complete Coverage Analysis. Check 1_Trimmed_Reads.\n";
}
system($cov_bowtie2_exec);
my $jellyfish_count_exec = $JELLYFISH . " count -m 25 -t ". $threads . " -C -s 1G map_*";
system($jellyfish_count_exec);
my $jellyfish_dump_exec = $JELLYFISH . " dump mer_counts.jf > " . $name . "_25dump";
system($jellyfish_dump_exec);
my $window_cov_exec = "perl " . $COVERAGE_DIR . "/new_window_coverage.pl " . $name . "_25dump ../Final_Assembly/" . $name . "_FULLCP.fsa 25";
system($window_cov_exec);
my $rscript_exec = "Rscript " . $COVERAGE_DIR . "/plot_coverage.r " . $name . ".coverage_25kmer.txt ". $name;
system($rscript_exec);
my $check_cov_exec;
if(defined $min_coverage){
$check_cov_exec = "perl " . $COVERAGE_DIR . "/check_plastid_coverage.pl " . $name . ".coverage_25kmer.txt 25 ".$min_coverage;
}
else{
$check_cov_exec = "perl " . $COVERAGE_DIR . "/check_plastid_coverage.pl " . $name . ".coverage_25kmer.txt 25";
}
my $coverage_used = `$check_cov_exec`;
chomp($coverage_used);
$current_runtime = localtime();
print $LOGFILE "$current_runtime\tMinimum coverage of $coverage_used for verifying assembly.\n";
print $SUMMARY "Minimum Coverage Used for Verification: $coverage_used\n";
chdir("../");
$current_runtime = localtime();
print $LOGFILE "$current_runtime\tCoverage analysis finished.\n";
if(-z "Coverage_Analysis/".$name."_problem_regions_plastid_assembly.txt"){
print $LOGFILE "\t\t\t\tNo issues with assembly coverage were identified.\n";
coverage_summary("Final_Assembly/$name\._CP_pieces.fsa", "Coverage_Analysis/");
if($clean){
if ($clean eq "light"){
unlink(glob("5_Plastome_Finishing/*fsa.n*"));
unlink(glob("*/*bt2"));
unlink(glob("Coverage_Analysis/*25dump"));
unlink(glob("Coverage_Analysis/mer_counts.jf"));
unlink(glob("*/*.sam"));
}
if ($clean eq "deep"){
rmdir("1_Trimmed_Reads");
rmdir("2_Bowtie_Mapping");
rmdir("3_Spades_Assembly");
rmdir("4_Afin_Assembly");
rmdir("5_Plastome_Finishing");
unlink(glob("Coverage_Analysis/*25dump"));
unlink(glob("Coverage_Analysis/mer_counts.jf"));
unlink(glob("*/*bt2"));
}
}
}
else{
print $LOGFILE "\t\t\t\tProblem areas identified for assembly coverage. Attempting to repair assembly.\n";
print $LOGFILE "********************STARTING REASSEMBLY********************\n";
print $SUMMARY "\nVALUES BELOW FROM REASSEMBLED PLASTOME\n";
mkdir("4.5_Reassemble_Low_Coverage");
chdir("4.5_Reassemble_Low_Coverage");
my $lc_remove_contigs = &reassemble_low_coverage("../Final_Assembly/" . $name . "_FULLCP.fsa", "../Coverage_Analysis/".$name."_problem_regions_plastid_assembly.txt");
$current_afin=&afin_wrap($lc_remove_contigs);
&orientate_plastome($current_afin, $name, "../Final_Assembly_Fixed_Low_Coverage/");
mkdir("../Coverage_Analysis_Reassembly");
chdir("../Coverage_Analysis_Reassembly");
my $build_bowtie2_exec = $BOWTIE2 . "-build ../Final_Assembly_Fixed_Low_Coverage/" . $name . "_FULLCP.fsa " . $name . "_bowtie";
system($build_bowtie2_exec);
my $cov_bowtie2_exec;
if(@p1_array){
$cov_bowtie2_exec = $BOWTIE2 . " --very-sensitive-local --quiet --al map_hits.fq --al-conc map_pair_hits.fq -p " . $threads . " -x " . $name ."_bowtie" . " -1 ../1_Trimmed_Reads/" . $name . ".trimmed_P1.fq -2 ../1_Trimmed_Reads/" . $name . ".trimmed_P2.fq -U ../1_Trimmed_Reads/" . $name . ".trimmed_UP.fq -S " . $name . ".sam";
}
else{
$cov_bowtie2_exec = $BOWTIE2 . " --very-sensitive-local --quiet --al map_hits.fq -p " . $threads . " -x " . $name ."_bowtie" . " -U ../1_Trimmed_Reads/" . $name . ".trimmed_UP.fq -S " . $name . ".sam";
}
system($cov_bowtie2_exec);
my $jellyfish_count_exec = $JELLYFISH . " count -m 25 -t ". $threads . " -C -s 1G map_*";
system($jellyfish_count_exec);