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PhyloFlash.pm
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PhyloFlash.pm
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use strict;
# see bottom of file for copyright & license
package PhyloFlash;
use Exporter qw(import);
use Time::Piece;
use Text::Wrap;
use Config;
use Storable;
$Text::Wrap::huge = "overflow";
=head1 NAME
PhyloFlash Perl module - functions for phyloFlash
=head1 SYNOPSIS
use PhyloFlash;
=head1 DESCRIPTION
This module contains helper functions shared by the phyloFlash scripts.
=head2 FUNCTIONS
=over
=cut
our $VERSION = "3.4.2";
our @ISA = qw(Exporter);
our @EXPORT = qw(
$VERSION
get_cpus
msg
@msg_log
write_logfile
err
version_sort
file_is_newer
get_subdirs
open_or_die
slurpfile
csv_escape
require_tools
check_environment
check_vsearch_version
run_prog
run_prog_nodie
file_download
fasta_copy_except
fasta_copy_iupac_randomize
cluster
hash2taxstring_counts
hashtreeconsensus
taxstring2hash
consensus_taxon_counter
revcomp_DNA
fix_hash_sortmerna_sam
split_sam_line_to_hash
initialize_outfiles_hash
);
use IPC::Cmd qw(can_run);
=item get_cpus ()
Returns the number of CPUs as listed in F</proc/cpuinfo> (Linux)
or reported by F<sysctl> (Darwin/OSX)
=cut
sub get_cpus {
if ($Config{"osname"} eq "linux") {
open(my $fh, "<", "/proc/cpuinfo") or return 1;
return scalar (map /^processor/, <$fh>)
}
elsif ($Config{"osname"} eq "darwin") {
can_run("sysctl") or return 1;
my $ncpu = `sysctl -n hw.ncpu`;
chomp($ncpu);
return $ncpu;
}
else {
return 1;
}
}
=item msg ($msg)
Logs a message to STDERR with time stamp prefix.
=cut
our @msg_log; # Global var to store log
sub msg {
my $t = localtime;
my $line = wrap ("[".$t->hms."] ", " ",
join "\n", @_);
push @msg_log, $line;
print STDERR $line."\n";
}
=item write_logfile ($file)
Saves message log to file
=cut
sub write_logfile {
my $file = shift;
msg ("Saving log to file $file");
my $fh;
open_or_die(\$fh, ">>", $file);
print $fh join "\n", @PhyloFlash::msg_log;
close($fh);
}
=item err($msg)
Logs an error message to STDERR and dies
=cut
sub err {
my @msg = (@_,"Aborting.");
$msg[0] = "FATAL: ".$msg[0];
msg(@msg);
write_logfile("phyloFlash_log_on_error");
exit(3);
}
=item version_sort(@paths)
sorts paths by descending alphanumeric order ignoring directories and
the part of the filename preceding the first digit. E.g.:
/home/xx/silva_125
/var/lib/dbs/silva_ssu_123.1
/software/phyloFlash_2.0/ssu_123
./121
=cut
sub version_sort {
# this will fail with Perl <5.13.2. Versions before that lack the
# non-destructive subsitition flag /r used in the map line below.
return map { $_->[0] }
sort { - ($a->[1] cmp $b->[1]) }
map { [ $_, ($_ =~ s/.*\/[^\d]*//r) =~ s[(\d+)][pack "N", $1]ger ] }
@_;
# works like this:
# last map creates a key->value map with the path the key and the value
# 1. having leading directories and filename up to first digit removed
# 2. all numbers converted to 4-byte strings (most significant first)
# sort does descending sort by string comparison
# first map picks just the key to return sorted array
}
=item file_is_newer ($file1, $file2)
Compares the time stamps of two files. Returns true if file1
is newer than file2.
=cut
sub file_is_newer {
my $file1 = shift;
my $file2 = shift;
return (stat($file1))[9] > (stat($file2))[9];
}
=item get_subdirs ($parent)
Returns array of paths to subdirs of $parent (hidden excluded).
=cut
sub get_subdirs {
my $parent = shift;
opendir(my $dh, $parent) or return ();
my @dirs = map { "$parent/" . $_} grep { !/^\./ && -d "$parent/$_" } readdir($dh);
closedir($dh);
return @dirs;
}
=item open_or_die (\$fh, $mode, $filename)
Opens a file handle and terminates with an error if the
open failed. $fh must be passed as reference!
=cut
sub open_or_die {
my ($fh, $mode, $fname) = @_;
my $msg;
if ($mode eq ">") {
$msg = "write to file";
} elsif ($mode eq ">>") {
$msg = "append to file";
} elsif ($mode eq "<") {
$msg = "read from file";
} elsif ($mode eq "-|") {
$msg = "run command";
}
open($$fh, $mode, $fname)
or err("Failed to $msg '$fname': $!");
}
=item slurpfile ($filename)
Opens a file, slurps contents into string, and returns string.
=cut
sub slurpfile {
my ($file) = @_;
my $return;
{
my $fh_slurp;
open_or_die(\$fh_slurp, "<", $file);
local $/ = undef;
$return = <$fh_slurp>;
close($fh_slurp);
}
return ($return);
}
=item csv_escape ($var)
Escapes data for CSV output according to RFC4180.
=cut
sub csv_escape {
return unless defined wantarray;
my @parms = @_;
for (@parms) {
if (m/[",\r\n]/) {
s/"/""/g;
$_='"'.$_.'"';
}
}
return wantarray ? @parms : $parms[0];
}
# we store a hash of tool_name=>tool_binary here
my %progs = (
vsearch => "vsearch"
);
=item require_tools (%hash)
Adds tools to the list of required tools. Argument must be
a hash. Use like so:
require_tools(("tool" => "mytool.pl"))
=cut
sub require_tools {
my %arg = @_;
%progs = (%progs, %arg);
}
=item check_environment ()
Verifies availability of all tools requested using require_tools().
The required tools are located using can_run and the paths determined
printed using msg(). If any of the tools are missing, this function
will abort, asking the user to fix the prerequisites.
=cut
sub check_environment {
my @missing;
msg("Checking for required tools.");
foreach my $prog (keys %progs) {
my $progname = $progs{$prog};
if ($progs{$prog} = can_run($progname)) {
msg("Using $prog found at \"".$progs{$prog}."\".");
} else {
push @missing, " $prog ($progname)";
}
}
if (@missing) {
msg("Unable to find all required tools. These are missing:");
foreach my $prog (@missing) {
msg($prog);
}
err("Please make sure these are installed and in your PATH.\n\n");
} else {
msg("All required tools found.");
}
}
=item check_vsearch_version ()
Check that Vsearch version is at least 2.5.0. Return "1" if yes, otherwise undef
UDB file is only implemented from 2.5.0 onwards
=cut
sub check_vsearch_version {
my $version_msg = `vsearch -v 2>&1`;
my $return_value;
if ($version_msg =~ m/vsearch v(\d+)\.(\d+)\.\d+/) {
my ($maj,$min) = ($1, $2); # Major and minor version
# Check if at least v2.5.0
if ($maj >= 2 && $min >= 5) {
$return_value = 1;
msg ("Vsearch v2.5.0+ found, will index database to UDB file");
}
} else {
msg ("Could not determine Vsearch version. Assuming to be < v2.5.0");
}
return $return_value;
}
=item run_prog ($progname, $args, $redir_stdout, $redir_sterr)
Runs a tool by the name given in require_tools.
Aborts the script if the tool returned with an error code.
=over 15
=item $progname
the name of the tool
=item $args
command line arguments to be passed
=item $redir_stdout
file or file descriptor to redirect stdout to
=item $redir_stdin
same for stderr
=back
=cut
sub run_prog {
my ($prog, $args, $redir_stdout, $redir_stderr) = @_;
if (not exists $progs{$prog}) {
msg("trying to launch unknown tool \"$prog\". pls add to %progs");
$progs{$prog} = can_run($prog) or err("Failed to find $prog");
}
my $cmd = $progs{$prog}." ".$args;
$cmd .= " >".$redir_stdout if ($redir_stdout);
$cmd .= " 2>".$redir_stderr if ($redir_stderr);
msg("running subcommand:","$cmd");
my $return = system($cmd);
if ($return != 0) {
my @errmsg = ("Tool execution failed!.",
"Error was '$!' and return code '$?'");
if ($redir_stdout) {
push @errmsg, "Check log file $redir_stdout";
}
if ($redir_stderr) {
push @errmsg, "Check error log file $redir_stderr";
}
err(@errmsg);
}
# FIXME: print tail of stderr if redirected
}
=item run_prog_nodie ($progname, $args, $redir_stdout, $redir_sterr)
Runs a tool by the name given in require_tools.
Does not abort the script if returned with error code
=over 15
=item $progname
the name of the tool
=item $args
command line arguments to be passed
=item $redir_stdout
file or file descriptor to redirect stdout to
=item $redir_stdin
same for stderr
=back
=cut
sub run_prog_nodie {
my ($prog, $args, $redir_stdout, $redir_stderr) = @_;
if (not exists $progs{$prog}) {
msg("trying to launch unknown tool \"$prog\". pls add to %progs");
$progs{$prog} = can_run($prog) or err("Failed to find $prog");
}
my $cmd = $progs{$prog}." ".$args;
$cmd .= " >".$redir_stdout if ($redir_stdout);
$cmd .= " 2>".$redir_stderr if ($redir_stderr);
msg("running subcommand:","$cmd");
my $return = system($cmd);
if ($return != 0) {
my @errmsg = ("Tool execution failed!.",
"Error was '$!' and return code '$?'");
if ($redir_stdout) {
push @errmsg, "Check log file $redir_stdout";
}
if ($redir_stderr) {
push @errmsg, "Check error log file $redir_stderr";
}
msg(@errmsg);
}
return ($return);
# FIXME: print tail of stderr if redirected
}
=item file_md5 ($filename)
Computes the (hex coded) MD5 sum for the contents of a local file ($filename).
=cut
sub file_md5 {
my $file = shift;
my $fh;
open($fh, "<", $file)
or err("Unable to open $file.");
my $ctx = Digest::MD5->new;
$ctx->addfile($fh);
close($fh);
return $ctx->hexdigest;
}
=item ftp_read_var ($ftp, $pattern)
Fetches the contents of a file from FTP. $ftp must be a connected
Net::FTP object and $pattern a file pattern relative to the
current path of $ftp. If the file exists, the contents are returned.
Otherwise returns an empty string
=cut
sub ftp_read_var {
my $ftp = shift;
my $file = shift;
my $out = "";
open(my $fh, '>', \$out);
$ftp->get($file, $fh);
close($fh);
return $out;
}
=item file_download ($path)
Downloads a file from a FTP server.
$path should be of the form "ftp.somedomain.tld/some/dirs/pa.*ttern".
Returns the name of the downloaded file.
=over 3
=item -
Compares byte sizes before and after download.
=item -
Compares MD5 sum if <file>.md5 exists on server.
=item -
File download is skipped if both size and MD5 (if exists) are correct
=item -
Asks user to confirm license if the directory containing the target
file also contains a file named LICENSE.txt.
=back
=cut
sub file_download {
my ($server,$path,$pat) = ($_[0] =~ /([^\/]*)(\/.*\/)([^\/]*)/);
msg(" Connecting to $server");
my $ftp = new Net::FTP($server,(
Passive => 1,
Debug => 0,
Timeout => 600
))
or err("Could not connect to $server");
$ftp->login("anonymous", "-phyloFlash")
or err("Could not login to $server:", $ftp->message);
$ftp->binary();
$ftp->pasv();
msg(" Finding $path$pat");
$ftp->cwd($path)
or err("Could not enter path '\$path\': ", $ftp->message);
my $files = $ftp->ls($pat)
or err("Could not list files matching \'$pat'\ in \'$path\': ",
$ftp->message);
err("No files found?!")
if (@$files == 0);
msg(" Multiple files found?! Using first of ".join(@$files))
if (@$files > 1);
my $file = shift(@$files);
my $file_size = $ftp->size($file)
or err("Could not get file size:", $ftp->message);
msg(" Found $file ($file_size bytes)");
# try downloading md5
my $file_md5 = ftp_read_var($ftp, $file.".md5");
$file_md5 =~ s/ .*//;
chomp($file_md5);
# compare with potentially existing file
if (-e $file) { # file exists
msg(" Found existing $file");
if (-s $file == $file_size) { # and has same size
if ($file_md5 eq "") {
msg("Sizes match. Skipping download");
return $file;
}
msg(" Verifying MD5...");
my $local_md5 = file_md5($file);
if ($file_md5 eq $local_md5) { # and has same md5
msg(" Verified. Skipping download");
return $file;
} else { # md5 mismatch
msg(" MD5 sum mismatch: '$file_md5' != '$local_md5'");
}
} else {
msg(" Size mismatch: ".$file_size." != ". -s $file);
}
msg(" -> re-downloading");
}
# verify license
my $license = ftp_read_var($ftp, "LICENSE.txt");
if (!$license eq "") {
msg("The file you are about to download comes with a license:\n\n\n"
.$license."\n\n");
msg("Do you wish to continue downloading under the conditions");
msg("specified above? [yes/no]: ");
my $accept;
while (<>) {
chomp;
if ($_ eq "yes") {
$accept = 1;
last;
} elsif ($_ eq "no") {
$accept = 0;
last;
} else {
msg("[yes/no]: ");
}
}
if ($accept == 0) {
msg("Ok. Goodbye...");
exit(0);
}
}
# download file
print STDERR "|" . "-" x 75 . "|\n";
$ftp->hash(\*STDERR, $ftp->size($file)/76);
$ftp->get($file)
or err("Failed to download $file:", $ftp->message);
print STDERR "\n";
err("File size mismatch?!")
if (-s $file != $file_size);
if ($file_md5 eq "") { # had no md5
return $file;
}
msg(" Verifying MD5...");
my $local_md5 = file_md5($file);
if ($local_md5 eq $file_md5) {
msg("File ok");
return $file;
}
err(" MD5 sum mismatch: '$file_md5' != '$local_md5'");
}
=item fasta_copy_except ($source, $dest, @accs)
Extracts all but the sequences listed in @accs from FASTA file
$source into FASTA file $dest.
=cut
sub fasta_copy_except {
my ($source, $dest, @accs, $overwrite) = @_;
my $sfh;
my $dfh;
if (! -e $dest || $overwrite == 1) { # Check whether file exists or overwrite allowed
open_or_die(\$sfh, "<", $source);
open_or_die(\$dfh, ">", $dest);
my %acc_hash = map { $_ => 1 } @accs;
my $skip = 0;
my $num_acc = scalar (keys %acc_hash);
msg ("Number of sequences to skip: $num_acc");
while(my $row = <$sfh>) {
if (substr($row, 0, 1) eq '>') {
my ($acc) = ($row =~ m/>([^ ]*) /);
$skip = exists $acc_hash{$acc};
}
print $dfh $row if (!$skip);
}
} else {
msg ("WARNING: File $dest already exists, will not overwrite");
}
}
=item cluster ($source, $dest, $id)
Runs vsearch's cluster_fast algorithm to extract centroids
from $source into $dest at a cluster size of $id.
=cut
sub cluster {
my ($src, $dst, $id, $cpus, $overwrite) = @_;
msg("clustering database");
if (! -e $dst || $overwrite == 1) {
run_prog("vsearch",
" --cluster_fast $src "
. "--id $id "
. "--centroids $dst "
. "--notrunclabels "
. "--threads $cpus ");
} else {
msg ("WARNING: File $dst already exists, not overwriting");
}
}
# hash of IUPAC characters coding for multiple possible bases
my %IUPAC_DECODE = (
"X" => "ACGT",
"R" => "AG",
"Y" => "CT",
"M" => "CA",
"K" => "TG",
"W" => "TA",
"S" => "CG",
"B" => "CTG",
"D" => "ATG",
"H" => "ATC",
"V" => "ACG",
"N" => "ACTG");
=item fasta_copy_iupac_randomize ($source, $dest)
Creates a normalized FASTA file $dest from FASTA file $source.
=over 3
=item -
removes alignment characters ("." and "-")
=item -
uppercases all bases
=item -
turns RNA into DNA (U->T)
=item -
replaces echo IUPAC coded ambiguous base with a base randomly chosen
from the set of options (i.e. replaces B with C, T or G).
=back
=cut
sub fasta_copy_iupac_randomize {
my ($infile, $outfile, $overwrite) = @_;
my ($ifh, $ofh);
if (! -e $outfile || $overwrite == 1) {
open_or_die(\$ifh, "<", $infile);
open_or_die(\$ofh, ">", $outfile);
# iterate over lines of FASTA file
while(my $row = <$ifh>) {
# pass through FASTA header
if (substr($row, 0, 1) eq ">") {
print $ofh $row;
next;
}
# remove alignment, uppercase, turn U into T
$row =~ tr/a-zA-Z.-/A-TTV-ZA-TTV-Z/d;
# split into ok / not-ok letter segments
for my $part ( split(/([^AGCT\n])/, $row) ) {
if (not $part =~ m/[AGCT\n]/) {
# segment in need of fix, iterate chars
for my $i ( 0 .. (length($part)-1) ) {
# get replacement character list
my $rpl = $IUPAC_DECODE{substr($part, $i, 1)};
substr($part, $i, 1) =
substr($rpl, rand(length($rpl)), 1)
if defined $rpl;
}
}
print $ofh $part;
}
}
} else {
msg ("WARNING: File $outfile already exists, not overwriting");
}
}
{
package Timer;
use strict;
use Time::Piece;
use Time::Seconds;
=item Timer->new ()
starts a new timer
=cut
sub new {
my ($class) = @_;
my $self = bless {}, $class;
$self->{time} = localtime;
return $self;
}
=item Timer->minutes ()
returns the runtime of the timer in minuts
=cut
sub minutes {
my ($self) = @_;
my $endtime = localtime;
my $diff = $endtime - $self->{time};
return sprintf "%.2f minutes", $diff->minutes;
}
}
=item hash2taxstring_counts
Collapse taxonomy tree into taxstrings and report counts. Taxa are encoded as
hash refs; counts as hash values.
=cut
sub hash2taxstring_counts {
my ($href, $taxstring, $href2) = @_;
foreach my $key (keys %$href) {
if (ref ($href->{$key}) eq 'HASH') { # Recursion
hash2taxstring_counts (\%{$href->{$key}}, "$taxstring;$key", $href2);
} else { # End condition - have reached a count
$href2->{"$taxstring;$key"} = $href->{$key};
}
}
}
=item hashtreeconsensus
Walk hash of taxonomy tree and report where it diverges from single branch.
As a side effect, report taxstring of the congruent portion of the tree
=cut
sub hashtreeconsensus {
my ($href, # Reference to hash of tax tree
$aref # Reference to array to store the congruent portion of tree
) = @_;
my @keys = keys %$href;
if (scalar @keys == 1) {
push @$aref, $keys[0];
if (ref($href->{$keys[0]}) eq 'HASH') { # Recursion
hashtreeconsensus(\%{$href->{$keys[0]}}, $aref);
} else {
return '1'; # The tree is identical to the tips
}
} else {
my @diverge = keys %$href;
return \@diverge; # Return the level at which the tree diverges
}
}
=item taxstring2hash
Convert taxonomy string to a nested hash structure recursively
=cut
sub taxstring2hash {
my ($href, # Reference to hash
$aref # Reference to taxonomy string as array
) = @_;
my $taxon = shift @$aref;
if (@$aref) { # Recursion
taxstring2hash (\%{$href->{$taxon}}, $aref);
} else { # End condition - count number of occurrences of this taxon
$href->{$taxon} ++;
}
}
=item consensus_taxon_counter
Take consensus portion of an array of taxon strings and hash into a taxonomy tree
=cut
sub consensus_taxon_counter {
my ($href_in, # Hash ref for taxonomy trees keyed by read
$aref, # Array ref of list of reads to summarize
$taxlevel, # Taxonomic level for summarizing
) = @_;
my %hashout;
my %taxhash;
foreach my $read (@$aref) {
next if (!defined $href_in->{$read}); # Skip if this read has no taxonomic assignment
# Get consensus taxstring for a given read
my @outarr;
my $return = hashtreeconsensus(\%{$href_in->{$read}}, \@outarr);
if (defined $return && @outarr) {
if (scalar @outarr > $taxlevel) {
# Trim to max taxonomic rank requested
@outarr = @outarr[0..$taxlevel-1];
} elsif (scalar @outarr < $taxlevel) {
# If taxonomic rank of consensus doesn't reach to requested rank,
# repeat the lowest taxonomic rank in brackets until requested
# rank, e.g. Bacteria;Proteobacteria;(Proteobacteria);(Proteobacteria); etc...
my $diff = $taxlevel - scalar (@outarr);
push @outarr, ("($outarr[$#outarr])") x $diff; # Parens before x operator make array
}
# Hash the consensus taxonomy into a taxonomic tree, with counts
# as end values for all reads
taxstring2hash(\%{$taxhash{'ROOT'}}, \@outarr);
}
}
my %taxcounts;
hash2taxstring_counts (\%taxhash, '', \%taxcounts);
foreach my $key (keys %taxcounts) { # Replace ugly ;ROOT; from taxstring to be displayed
my $display_taxstring = $key;
$display_taxstring =~ s/^;ROOT;//;
$hashout{$display_taxstring} = $taxcounts{$key};
}
return \%hashout;
}
=item revcomp_DNA ($seq)
Return reverse complement of a DNA sequence
=cut
sub revcomp_DNA {
my ($seq) = @_;
my $rev = reverse $seq;
$rev =~ tr/ATCGatcgYRWSKMDVHByrwskmdvhb/TAGCtagcRYWSMKHBDVrywsmkhbdv/; # Include ambiguity codes
return $rev;
}
=item fix_hash_sortmerna_sam
Fix SAM file produced by Sortmerna. Returns ref to array of fixed SAM file lines
=cut
sub fix_hash_sortmerna_sam {
# Fix SAM file produced by Sortmerna v2.1b
my ($fastq,
$sam_original,
$acc2tax,
$semode) = @_;
my $pe;
if ($semode == 0) {
$pe = 1;
} else {
$pe = 0;
}
# Read in Fastq file and hash read orientations and sequences
my $fastq_href = read_interleaved_fastq ($fastq);
#print Dumper $fastq_href;
# Read in SAM file and fix bit flags 0x1, 0x40, 0x80, 0x100
my ($sambyread_href, $sambyline_aref) = read_sortmerna_sam($sam_original, $fastq_href, $pe);
# Fix flags related to read pairs: 0x2, 0x4, 0x8, 0x20
fix_pairing_flags($sambyread_href, $fastq_href) if $pe == 1;
#print Dumper $sambyread_href;
# Add back the tax strings to RNAME field
if (defined $acc2tax) {
my $acc2tax_href = retrieve ($acc2tax);
fix_rname_taxstr($sambyread_href, $acc2tax_href);
}
# Flatten hash back to array and print to output
my $aref = samaref_to_lines($sambyline_aref, $fastq_href);
#foreach my $line (@$aref) {
# print "$line\n";
#}
# Return ref to array containing the fixed SAM file including spliced
# dummy sequences for unmapped segments
# Return also sambyread_href and sambyline_aref
return ($sambyread_href,
$sambyline_aref,
$aref,
);
}
sub fix_rname_taxstr {
# Function used by fix_hash_sortmerna_sam
# Replace the accession number of RNAME with the original version
# retrieved from hash of acc vs taxstring
my ($sam_href,
$acc_href
) = @_;
foreach my $id (keys %$sam_href) {
foreach my $segment (keys %{$sam_href->{$id}}) {
foreach my $rname (keys %{$sam_href->{$id}{$segment}}) {
foreach my $href (@{$sam_href->{$id}{$segment}{$rname}}) {
my $new_rname = join " ", ($href->{'RNAME'}, $acc_href->{$href->{'RNAME'}});
$href->{'RNAME'} = $new_rname;
}
}
}
}
}
sub samaref_to_lines {
# Function used by fix_hash_sortmerna_sam
# Reconstruct SAM lines, and also insert dummy entries for unmapped read fwd segments
# Input is an array of hash references and hash of Fastq sequences produced
# by read_interleaved_fastq()
my ($aref,
$fastq_href) = @_;
my @lines;
foreach my $href (@$aref) {
# Split read ID
my $id_fwd = $href->{'QNAME'};
#my $id_rev = $href->{'QNAME'};
my $sep;
if ($id_fwd =~ m/^(.+)([:\/_])[12]$/) {
$id_fwd = $1.$2.'1';
#$id_rev = $1.$2.'2';
}
# Splice in first segment dummy entry if this is rev with fwd read unmapped
if ($href->{'FLAG'} & 0x80 && $href->{'FLAG'} & 0x8) {
my @splice = ($id_fwd, # QNAME
0x1 + 0x4 + 0x40, # Paired read, segment unmapped, first segment
'*', # RNAME
'0', # POS
'255', # MAPQ
'*', # CIGAR
'*', # RNEXT
'0', # PNEXT
'0', # TLEN
$fastq_href->{$href->{'QNAME'}}{'fwd'}{'seq'}, # SEQ
$fastq_href->{$href->{'QNAME'}}{'fwd'}{'qual'} # QUAL
);
push @lines, join "\t", @splice unless $href->{'FLAG'} & 0x100;
}
# Otherwise continue
my @outarr;
my @fields = qw(QNAME FLAG RNAME POS MAPQ CIGAR RNEXT PNEXT TLEN SEQ QUAL);
foreach my $field (@fields) {