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clfilterseq.pl
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use strict;
use Fcntl ':flock';
use File::Spec;
my $buildno = '0.9.x';
# options
my $append;
my $minlen = 1;
my $maxlen;
my $ovlen = 'truncate';
my $minqual;
my $maxqual;
my $ovqual = 'cap';
my $minquallen;
my $minmeanqual;
my $maxplowqual;
my $replaceinternal;
my %keywords;
my %ngwords;
my $converse;
my $minnseq;
my $folder;
my $runname;
my $minnreplicate = 2;
my $minpreplicate = 1;
my $minnpositive = 1;
my $minppositive = 0;
my $numthreads = 1;
my $targetrank;
my $numbering = 1;
my $fuseotu = 1;
my $topN;
# Input/Output
my @inputfiles;
my $output;
my $otufile;
my $replicatelist;
my $taxfile;
my $otulist;
my $otuseq;
my $notulist;
my $notuseq;
# other variables
my $devnull = File::Spec->devnull();
my $format;
my %members;
my %eliminate;
my @taxrank = ('no rank', 'superkingdom', 'kingdom', 'subkingdom', 'superphylum', 'phylum', 'subphylum', 'superclass', 'class', 'subclass', 'infraclass', 'cohort', 'subcohort', 'superorder', 'order', 'suborder', 'infraorder', 'parvorder', 'superfamily', 'family', 'subfamily', 'tribe', 'subtribe', 'genus', 'subgenus', 'section', 'subsection', 'series', 'species group', 'species subgroup', 'species', 'subspecies', 'varietas', 'forma', 'forma specialis', 'strain', 'isolate');
my %taxrank;
for (my $i = 0; $i < scalar(@taxrank); $i ++) {
$taxrank{$taxrank[$i]} = $i;
}
my %taxonomy;
my %otu2newotu;
my %newotu2otu;
my %otulist;
my %notulist;
my $qthreads;
my $hthreads;
# file handles
my $filehandleinput1;
my $filehandleinput2;
my $filehandleoutput1;
&main();
sub main {
# print startup messages
&printStartupMessage();
# get command line arguments
&getOptions();
# check variable consistency
&checkVariables();
# read list files
&readListFiles();
# read sequence files
&readSequenceFiles();
# read taxonomy file
&readTaxonomyFile();
# read otufile
&readMembers();
# recognize file format
&recognizeFormat();
# process sequences
&processSequences();
# concatenate FASTQ files
&concatenateFiles();
}
sub printStartupMessage {
print(STDERR <<"_END");
clfilterseq $buildno
=======================================================================
Official web site of this script is
https://www.fifthdimension.jp/products/claident/ .
To know script details, see above URL.
Copyright (C) 2011-XXXX Akifumi S. Tanabe
This program is free software; you can redistribute it and/or modify
it under the terms of the GNU General Public License as published by
the Free Software Foundation; either version 2 of the License.
This program is distributed in the hope that it will be useful,
but WITHOUT ANY WARRANTY; without even the implied warranty of
MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the
GNU General Public License for more details.
You should have received a copy of the GNU General Public License along
with this program; if not, write to the Free Software Foundation, Inc.,
51 Franklin Street, Fifth Floor, Boston, MA 02110-1301 USA.
_END
# display usage if command line options were not specified
unless (@ARGV) {
&helpMessage();
}
}
sub getOptions {
$output = $ARGV[-1];
my %inputfiles;
for (my $i = 0; $i < scalar(@ARGV) - 1; $i ++) {
if ($ARGV[$i] =~ /^-+(?:keyword|keywords)=(.+)$/i) {
my $keywords = $1;
foreach my $keyword (split(/,/, $keywords)) {
$keywords{$keyword} = 1;
}
}
elsif ($ARGV[$i] =~ /^-+(?:ngword|ngwords)=(.+)$/i) {
my $ngwords = $1;
foreach my $ngword (split(/,/, $ngwords)) {
$ngwords{$ngword} = 1;
}
}
elsif ($ARGV[$i] =~ /^-+otu=(.+)$/i) {
my @temp = split(',', $1);
foreach my $temp (@temp) {
$otulist{$temp} = 1;
}
}
elsif ($ARGV[$i] =~ /^-+n(?:egative)?otu=(.+)$/i) {
my @temp = split(',', $1);
foreach my $temp (@temp) {
$notulist{$temp} = 1;
}
}
elsif ($ARGV[$i] =~ /^-+otulist=(.+)$/i) {
$otulist = $1;
}
elsif ($ARGV[$i] =~ /^-+n(?:egative)?otulist=(.+)$/i) {
$notulist = $1;
}
elsif ($ARGV[$i] =~ /^-+otuseq=(.+)$/i) {
$otuseq = $1;
}
elsif ($ARGV[$i] =~ /^-+n(?:egative)?otuseq=(.+)$/i) {
$notuseq = $1;
}
elsif ($ARGV[$i] =~ /^-+(?:c|converse)$/i) {
$converse = 1;
}
elsif ($ARGV[$i] =~ /^-+folder$/i) {
$folder = 1;
}
elsif ($ARGV[$i] =~ /^-+(?:o|output)=(?:folder|dir|directory)$/i) {
$folder = 1;
}
elsif ($ARGV[$i] =~ /^-+otufile=(.+)$/i) {
$otufile = $1;
}
elsif ($ARGV[$i] =~ /^-+min(?:imum)?len(?:gth)?=(\d+)$/i) {
$minlen = $1;
}
elsif ($ARGV[$i] =~ /^-+max(?:imum)?len(?:gth)?=(\d+)$/i) {
$maxlen = $1;
}
elsif ($ARGV[$i] =~ /^-+(?:overflow|over|ov)(?:len|length)=(.+)$/i) {
my $value = $1;
if ($value =~ /^(?:elim|eliminate)$/i) {
$ovlen = 'eliminate';
}
elsif ($value =~ /^(?:trunc|truncate)$/i) {
$ovlen = 'truncate';
}
else {
&errorMessage(__LINE__, "\"$ARGV[$i]\" is invalid option.");
}
}
elsif ($ARGV[$i] =~ /^-+min(?:imum)?qual(?:ity)?=(\d+)$/i) {
$minqual = $1;
}
elsif ($ARGV[$i] =~ /^-+max(?:imum)?qual(?:ity)?=(\d+)$/i) {
$maxqual = $1;
}
elsif ($ARGV[$i] =~ /^-+(?:over|ov)qual(?:ity)?=(.+)$/i) {
my $value = $1;
if ($value =~ /^(?:elim|eliminate)$/i) {
$ovqual = 'eliminate';
}
elsif ($value =~ /^cap$/i) {
$ovqual = 'cap';
}
else {
&errorMessage(__LINE__, "\"$ARGV[$i]\" is invalid option.");
}
}
elsif ($ARGV[$i] =~ /^-+min(?:imum)?qual(?:ity)?len(?:gth)?=(\d+)$/i) {
$minquallen = $1;
}
elsif ($ARGV[$i] =~ /^-+min(?:imum)?meanqual(?:ity)?=(\d+)$/i) {
$minmeanqual = $1;
}
elsif ($ARGV[$i] =~ /^-+max(?:imum)?(?:r|rate|p|percentage)lowqual(?:ity)?=(.+)$/i) {
$maxplowqual = $1;
}
elsif ($ARGV[$i] =~ /^-+min(?:imum)?(?:n|num)(?:seqs?|sequences?|reads?)=(\d+)$/i) {
$minnseq = $1;
}
elsif ($ARGV[$i] =~ /^-+replaceinternal$/i) {
$replaceinternal = 1;
}
elsif ($ARGV[$i] =~ /^-+(?:replicate|repl?)list=(.+)$/i) {
$replicatelist = $1;
}
elsif ($ARGV[$i] =~ /^-+min(?:imum)?n(?:um)?(?:replicate|repl?)=(\d+)$/i) {
$minnreplicate = $1;
}
elsif ($ARGV[$i] =~ /^-+min(?:imum)?(?:r|rate|p|percentage)(?:replicate|repl?)=(\d(?:\.\d+)?)$/i) {
$minpreplicate = $1;
}
elsif ($ARGV[$i] =~ /^-+min(?:imum)?n(?:um)?positive=(\d+)$/i) {
$minnpositive = $1;
}
elsif ($ARGV[$i] =~ /^-+min(?:imum)?(?:r|rate|p|percentage)positive=(\d(?:\.\d+)?)$/i) {
$minppositive = $1;
}
elsif ($ARGV[$i] =~ /^-+(?:a|append)$/i) {
$append = 1;
}
elsif ($ARGV[$i] =~ /^-+(?:n|n(?:um)?threads?)=(\d+)$/i) {
$numthreads = $1;
}
elsif ($ARGV[$i] =~ /^-+runname=(.+)$/i) {
$runname = $1;
}
elsif ($ARGV[$i] =~ /^-+(?:tax|taxonomy)file=(.+)$/i) {
$taxfile = $1;
}
elsif ($ARGV[$i] =~ /^-+(?:target)?(?:tax|taxonomy)?(?:unit|rank|level)=(.+)$/i) {
my $taxrank = $1;
if ($taxrank{$taxrank}) {
$targetrank = $taxrank{$taxrank};
}
else {
&errorMessage(__LINE__, "\"$ARGV[$i]\" is invalid option.");
}
}
elsif ($ARGV[$i] =~ /^-+numbering=(.+)$/i) {
my $value = $1;
if ($value =~ /^(?:enable|e|yes|y|true|t)$/i) {
$numbering = 1;
}
elsif ($value =~ /^(?:disable|d|no|n|false|f)$/i) {
$numbering = 0;
}
else {
&errorMessage(__LINE__, "\"$ARGV[$i]\" is invalid option.");
}
}
elsif ($ARGV[$i] =~ /^-+fuseotu=(.+)$/i) {
my $value = $1;
if ($value =~ /^(?:enable|e|yes|y|true|t)$/i) {
$fuseotu = 1;
}
elsif ($value =~ /^(?:disable|d|no|n|false|f)$/i) {
$fuseotu = 0;
}
else {
&errorMessage(__LINE__, "\"$ARGV[$i]\" is invalid option.");
}
}
elsif ($ARGV[$i] =~ /^-+topN=(\d+)$/i) {
$topN = $1;
}
else {
my @temp = glob($ARGV[$i]);
if (scalar(@temp) > 0) {
foreach (@temp) {
if (!exists($inputfiles{$_})) {
$inputfiles{$_} = 1;
push(@inputfiles, $_);
}
else {
&errorMessage(__LINE__, "\"$_\" is doubly specified.");
}
}
}
else {
&errorMessage(__LINE__, "Input file does not exist.");
}
}
}
}
sub checkVariables {
if (scalar(@inputfiles) < 1) {
&errorMessage(__LINE__, "Input file was not given.");
}
if (scalar(@inputfiles) > 2) {
&errorMessage(__LINE__, "Too many input files were given.");
}
if (scalar(@inputfiles) == 2) {
$folder = 1;
}
if (-e $output && !$append) {
&errorMessage(__LINE__, "\"$output\" already exists.");
}
elsif ($folder && !mkdir($output) && !$append) {
&errorMessage(__LINE__, 'Cannot make output folder.');
}
else {
my @temp = glob("$output.*");
if (@temp) {
&errorMessage(__LINE__, "Temporary files already exist.");
}
}
if ($otufile && !-e $otufile) {
&errorMessage(__LINE__, "\"$otufile\" does not exist.");
}
if ($otulist && !-e $otulist) {
&errorMessage(__LINE__, "\"$otulist\" does not exist.");
}
if ($notulist && !-e $notulist) {
&errorMessage(__LINE__, "\"$notulist\" does not exist.");
}
if ($otuseq && !-e $otuseq) {
&errorMessage(__LINE__, "\"$otuseq\" does not exist.");
}
if ($notuseq && !-e $notuseq) {
&errorMessage(__LINE__, "\"$notuseq\" does not exist.");
}
if ($otulist && $notulist) {
&errorMessage(__LINE__, "The OTU list and the negative OTU list cannot be given at the same time.");
}
if ($otulist && $notuseq) {
&errorMessage(__LINE__, "The OTU list and the negative OTU sequence cannot be given at the same time.");
}
if ($otuseq && $notulist) {
&errorMessage(__LINE__, "The OTU sequence and the negative OTU list cannot be given at the same time.");
}
if ($otuseq && $notuseq) {
&errorMessage(__LINE__, "The OTU sequence and the negative OTU sequence cannot be given at the same time.");
}
if (%otulist && %notulist) {
&errorMessage(__LINE__, "The OTU list and the negative OTU list cannot be given at the same time.");
}
if ($replicatelist && !-e $replicatelist) {
&errorMessage(__LINE__, "\"$replicatelist\" does not exist.");
}
if ($taxfile && !-e $taxfile) {
&errorMessage(__LINE__, "\"$taxfile\" does not exist.");
}
if (!$targetrank) {
$targetrank = $taxrank{'species'};
}
if (($minnseq || $replicatelist) && !$otufile) {
my $prefix = $inputfiles[0];
$prefix =~ s/\.(?:gz|bz2|xz)$//i;
$prefix =~ s/\.[^\.]+$//;
if (-e "$prefix.otu.gz") {
$otufile = "$prefix.otu.gz";
}
elsif (-e "$prefix.otu") {
$otufile = "$prefix.otu";
}
}
if ($minnseq && !$otufile) {
&errorMessage(__LINE__, "The minimum number threshold for reads of contigs requires OTU file.");
}
if ($replicatelist && !$otufile) {
&errorMessage(__LINE__, "Replicate list requires OTU file.");
}
if ($minnseq && $replicatelist) {
&errorMessage(__LINE__, "The minimum number threshold for reads of contigs is incompatible to replicate list.");
}
if ($minnreplicate < 2) {
&errorMessage(__LINE__, "The minimum number of replicate is invalid.");
}
if ($minpreplicate > 1) {
&errorMessage(__LINE__, "The minimum percentage of replicate is invalid.");
}
if ($minppositive > 1) {
&errorMessage(__LINE__, "The minimum percentage of true positive for noisy/chimeric OTU detection is invalid.");
}
if ($minlen < 1) {
&errorMessage(__LINE__, "Minimum length must be equal to or more than 1.");
}
if ($minqual) {
$minqual += 33;
}
if ($minmeanqual) {
$minmeanqual += 33;
}
$qthreads = int($numthreads / 4);
if ($qthreads < 1) {
$qthreads = 1;
}
$hthreads = int($numthreads / 2);
if ($hthreads < 1) {
$hthreads = 1;
}
}
sub readListFiles {
if ($otulist) {
foreach my $otu (&readList($otulist)) {
$otulist{$otu} = 1;
}
}
elsif ($notulist) {
foreach my $otu (&readList($notulist)) {
$notulist{$otu} = 1;
}
}
}
sub readSequenceFiles {
if ($otuseq) {
foreach my $otu (&readSeq($otuseq)) {
$otulist{$otu} = 1;
}
}
elsif ($notuseq) {
foreach my $otu (&readSeq($notuseq)) {
$notulist{$otu} = 1;
}
}
}
sub readTaxonomyFile {
if ($taxfile) {
my @rank;
unless (open($filehandleinput1, "< $taxfile")) {
&errorMessage(__LINE__, "Cannot read \"$taxfile\".");
}
{
my $lineno = 1;
while (<$filehandleinput1>) {
s/\r?\n?$//;
if ($lineno == 1 && /\t/) {
@rank = split(/\t/, lc($_));
shift(@rank);
foreach my $rank (@rank) {
if (!exists($taxrank{$rank})) {
&errorMessage(__LINE__, "\"$rank\" is invalid taxonomic rank.");
}
}
}
elsif (/\t/) {
my @entry = split(/\t/, $_, -1);
my $otuname = shift(@entry);
if (scalar(@entry) == scalar(@rank)) {
for (my $i = 0; $i < scalar(@entry); $i ++) {
$taxonomy{$otuname}{$taxrank{$rank[$i]}} = $entry[$i];
}
}
else {
&errorMessage(__LINE__, "Input taxonomy file is invalid.");
}
}
else {
&errorMessage(__LINE__, "Input taxonomy file is invalid.");
}
$lineno ++;
}
}
close($filehandleinput1);
}
}
sub readMembers {
# read OTU file
if ($otufile && ($replicatelist || $taxfile)) {
# read OTU file and store
my %table;
my %otusum;
my @otunames;
$filehandleinput1 = &readFile($otufile);
my $otuname;
while (<$filehandleinput1>) {
s/\r?\n?$//;
s/;+size=\d+;*//g;
if (/^>(.+)$/) {
$otuname = $1;
push(@otunames, $otuname);
}
elsif ($otuname && / SN:(\S+)/) {
my $samplename = $1;
my @temp = split(/__/, $samplename);
if (scalar(@temp) == 3) {
my ($temprunname, $tag, $primer) = @temp;
if ($runname) {
$temprunname = $runname;
}
$table{"$temprunname\__$tag\__$primer"}{$otuname} ++;
$otusum{$otuname} ++;
}
else {
&errorMessage(__LINE__, "\"$_\" is invalid name.");
}
}
else {
&errorMessage(__LINE__, "\"$otufile\" is invalid.");
}
}
close($filehandleinput1);
if ($replicatelist) {
# read replicatelist
my %replicate;
$filehandleinput1 = &readFile($replicatelist);
while (<$filehandleinput1>) {
s/\r?\n?$//;
my @temp = split(/\t/, $_);
for (my $i = 0; $i < scalar(@temp); $i ++) {
push(@{$replicate{$temp[0]}}, $temp[$i]);
}
}
close($filehandleinput1);
# determine whether chimeric OTU or not within sample
my %withinsample;
foreach my $sample (keys(%replicate)) {
my %nreps;
my %nreads;
foreach my $replicate (@{$replicate{$sample}}) {
foreach my $otuname (@otunames) {
if ($table{$replicate}{$otuname}) {
$nreps{$otuname} ++;
$nreads{$otuname} += $table{$replicate}{$otuname};
}
}
}
foreach my $otuname (keys(%nreps)) {
if ($nreps{$otuname} < $minnreplicate || ($nreps{$otuname} / @{$replicate{$sample}}) < $minpreplicate) {
$withinsample{$otuname} += $nreads{$otuname};
}
}
}
# determine whether chimeric OTU or not in total
foreach my $otuname (@otunames) {
if ($withinsample{$otuname} >= $minnpositive && ($withinsample{$otuname} / $otusum{$otuname}) >= $minppositive) {
$eliminate{$otuname} = 1;
}
}
}
if ($taxfile) {
my %newotu;
foreach my $otuname (@otunames) {
if ($taxonomy{$otuname}{$targetrank}) {
my $taxon = $taxonomy{$otuname}{$targetrank};
$taxon =~ s/ /_/g;
unless ($fuseotu) {
$taxon = $otuname . ':' . $taxon;
}
$otu2newotu{$otuname} = $taxon;
$newotu{$taxon} = 0;
}
}
if (%otu2newotu) {
foreach my $samplename (keys(%table)) {
foreach my $otuname (@otunames) {
if ($taxonomy{$otuname}{$targetrank}) {
$newotu{$otu2newotu{$otuname}} += $table{$samplename}{$otuname};
if (!defined($newotu2otu{$otu2newotu{$otuname}}) || $table{$samplename}{$otuname} > $table{$samplename}{$newotu2otu{$otu2newotu{$otuname}}}) {
$newotu2otu{$otu2newotu{$otuname}} = $otuname;
}
}
}
}
}
@otunames = sort({$newotu{$b} <=> $newotu{$a}} keys(%newotu));
if ($topN) {
while (scalar(@otunames) > $topN) {
my $otuname = pop(@otunames);
delete($newotu2otu{$otuname});
}
}
if ($numbering) {
my $length = length(scalar(@otunames));
my $num = 1;
foreach my $otuname (@otunames) {
my $newotu = sprintf("%0*d", $length, $num) . "_$otuname";
$newotu2otu{$newotu} = $newotu2otu{$otuname};
delete($newotu2otu{$otuname});
$num ++;
}
}
}
}
elsif ($otufile && $minnseq) {
$filehandleinput1 = &readFile($otufile);
my $otuname;
while (<$filehandleinput1>) {
s/\r?\n?$//;
s/;+size=\d+;*//g;
if (/^>(.+)$/) {
$otuname = $1;
}
elsif ($otuname && /^([^>].*)$/) {
$members{$otuname} ++;
}
else {
&errorMessage(__LINE__, "\"$otufile\" is invalid.");
}
}
close($filehandleinput1);
}
}
sub recognizeFormat {
$filehandleinput1 = &readFile($inputfiles[0]);
while (<$filehandleinput1>) {
if (/^\@/) {
$format = 'FASTQ';
last;
}
elsif (/^>/) {
$format = 'FASTA';
last;
}
}
close($filehandleinput1);
unless ($format) {
&errorMessage(__LINE__, "Cannot recognize format.");
}
}
sub processSequences {
print(STDERR "Processing sequences...\n");
$filehandleinput1 = &readFile($inputfiles[0]);
if ($inputfiles[1]) {
$filehandleinput2 = &readFile($inputfiles[1]);
}
if ($format eq 'FASTQ') {
my $tempnline = 1;
my $seqname1;
my $nucseq1;
my $qualseq1;
my $seqname2;
my $nucseq2;
my $qualseq2;
my %pid;
my $child = 0;
my $nchild = 1;
$| = 1;
$? = 0;
# Processing FASTQ in parallel
while (<$filehandleinput1>) {
s/\r?\n?$//;
if ($tempnline % 4 == 1 && /^\@(.+)/) {
$seqname1 = $1;
$seqname1 =~ s/;+size=\d+;*//g;
if ($filehandleinput2) {
$seqname2 = readline($filehandleinput2);
$seqname2 =~ s/^\@//;
$seqname2 =~ s/^\s*\r?\n?$//;
$seqname2 =~ s/;+size=\d+;*//g;
}
}
elsif ($tempnline % 4 == 2) {
s/[^a-zA-Z]//g;
$nucseq1 = uc($_);
if ($filehandleinput2) {
$nucseq2 = readline($filehandleinput2);
$nucseq2 =~ s/\r?\n?$//;
$nucseq2 =~ s/[^a-zA-Z]//g;
$nucseq2 = uc($nucseq2);
}
}
elsif ($tempnline % 4 == 3 && /^\+/) {
$tempnline ++;
if ($filehandleinput2) {
readline($filehandleinput2);
}
next;
}
elsif ($tempnline % 4 == 0 && $seqname1) {
s/\s//g;
$qualseq1 = $_;
if ($filehandleinput2) {
$qualseq2 = readline($filehandleinput2);
$qualseq2 =~ s/\r?\n?$//;
$qualseq2 =~ s/\s//g;
$qualseq2 = $qualseq2;
}
if (my $pid = fork()) {
$pid{$pid} = $child;
if ($nchild == $numthreads * 2) {
my $endpid = wait();
while (!exists($pid{$endpid}) && $endpid != -1) {
$endpid = wait();
}
if (exists($pid{$endpid})) {
$child = $pid{$endpid};
delete($pid{$endpid});
}
elsif ($endpid == -1) {
$child = 0;
}
}
elsif ($nchild < $numthreads * 2) {
$child = $nchild;
$nchild ++;
}
if ($?) {
&errorMessage(__LINE__);
}
undef($seqname1);
undef($nucseq1);
undef($qualseq1);
undef($seqname2);
undef($nucseq2);
undef($qualseq2);
$tempnline ++;
next;
}
else {
if (!$eliminate{$seqname1}) {
if (scalar(@inputfiles) == 2 && $nucseq1 && $qualseq1 && $nucseq2 && $qualseq2) {
($nucseq1, $qualseq1) = &processOneSequence($seqname1, $nucseq1, $qualseq1);
($nucseq2, $qualseq2) = &processOneSequence($seqname2, $nucseq2, $qualseq2);
if ($nucseq1 && $qualseq1 && $nucseq2 && $qualseq2) {
{
my $filename = $inputfiles[0];
$filename =~ s/^.+[\\\/]//;
if ($taxfile) {
&saveToFile($nucseq1, $qualseq1, $otu2newotu{$seqname1}, $filename, $child);
}
else {
&saveToFile($nucseq1, $qualseq1, $seqname1, $filename, $child);
}
}
{
my $filename = $inputfiles[1];
$filename =~ s/^.+[\\\/]//;
if ($taxfile) {
&saveToFile($nucseq2, $qualseq2, $otu2newotu{$seqname2}, $filename, $child);
}
else {
&saveToFile($nucseq2, $qualseq2, $seqname2, $filename, $child);
}
}
}
}
elsif (scalar(@inputfiles) == 1 && $nucseq1 && $qualseq1 && !$nucseq2 && !$qualseq2) {
($nucseq1, $qualseq1) = &processOneSequence($seqname1, $nucseq1, $qualseq1);
if ($nucseq1 && $qualseq1) {
my $filename = $inputfiles[0];
$filename =~ s/^.+[\\\/]//;
if ($taxfile) {
&saveToFile($nucseq1, $qualseq1, $otu2newotu{$seqname1}, $filename, $child);
}
else {
&saveToFile($nucseq1, $qualseq1, $seqname1, $filename, $child);
}
}
}
}
exit;
}
}
else {
&errorMessage(__LINE__, "Invalid FASTQ.\nFile: $inputfiles[0]\nLine: $tempnline");
}
$tempnline ++;
}
# join
while (wait != -1) {
if ($?) {
&errorMessage(__LINE__, 'Cannot split sequence file correctly.');
}
}
}
elsif ($format eq 'FASTA') {
my $seqname1;
my $nucseq1;
my $seqname2;
my $nucseq2;
my $temp;
my %pid;
my $child = 0;
my $nchild = 1;
$| = 1;
$? = 0;
local $/ = "\n>";
while (<$filehandleinput1>) {
if (/^>?\s*(\S[^\r\n]*)\r?\n(.*)/s) {
$seqname1 = $1;
$nucseq1 = uc($2);
$seqname1 =~ s/;+size=\d+;*//g;
$nucseq1 =~ s/[^A-Z]//g;
if ($nucseq1) {
if ($filehandleinput2) {
$temp = readline($filehandleinput2);
$temp =~ /^>?\s*(\S[^\r\n]*)\r?\n(.*)\r?\n?/s;
$seqname2 = $1;
$nucseq2 = uc($2);
$seqname2 =~ s/;+size=\d+;*//g;
$nucseq2 =~ s/[^A-Z]//g;
}
if (my $pid = fork()) {
$pid{$pid} = $child;
if ($nchild == $numthreads * 2) {
my $endpid = wait();
while (!exists($pid{$endpid}) && $endpid != -1) {
$endpid = wait();
}
if (exists($pid{$endpid})) {
$child = $pid{$endpid};
delete($pid{$endpid});
}
elsif ($endpid == -1) {
$child = 0;
}
}
elsif ($nchild < $numthreads * 2) {
$child = $nchild;
$nchild ++;
}
if ($?) {
&errorMessage(__LINE__);
}
undef($seqname1);
undef($nucseq1);
undef($seqname2);
undef($nucseq2);
next;
}
else {
if (!$eliminate{$seqname1}) {
if (scalar(@inputfiles) == 2 && $nucseq1 && $nucseq2) {
$nucseq1 = &processOneSequence($seqname1, $nucseq1);
$nucseq2 = &processOneSequence($seqname2, $nucseq2);
if ($nucseq1 && $nucseq2) {
{
my $filename = $inputfiles[0];
$filename =~ s/^.+[\\\/]//;
if ($taxfile) {
&saveToFile($nucseq1, '', $otu2newotu{$seqname1}, $filename, $child);
}
else {
&saveToFile($nucseq1, '', $seqname1, $filename, $child);
}
}
{
my $filename = $inputfiles[1];
$filename =~ s/^.+[\\\/]//;
if ($taxfile) {
&saveToFile($nucseq2, '', $otu2newotu{$seqname2}, $filename, $child);
}
else {
&saveToFile($nucseq2, '', $seqname2, $filename, $child);
}
}
}
}
elsif (scalar(@inputfiles) == 1 && $nucseq1) {
$nucseq1 = &processOneSequence($seqname1, $nucseq1);
if ($nucseq1) {
my $filename = $inputfiles[0];
$filename =~ s/^.+[\\\/]//;
if ($taxfile) {
&saveToFile($nucseq1, '', $otu2newotu{$seqname1}, $filename, $child);
}
else {
&saveToFile($nucseq1, '', $seqname1, $filename, $child);
}
}
}
}
exit;
}
}
}
}
# join
while (wait != -1) {
if ($?) {
&errorMessage(__LINE__, 'Cannot split sequence file correctly.');
}
}
}
close($filehandleinput1);
if ($filehandleinput2) {
close($filehandleinput2);
}
print(STDERR "done.\n\n");
}
sub concatenateFiles {
print(STDERR "Concatenating files...\n");
{
my $child = 0;
$| = 1;
$? = 0;
foreach my $inputfile (@inputfiles) {
if (my $pid = fork()) {
$child ++;
if ($child == 4) {
if (wait == -1) {
$child = 0;
} else {
$child --;
}
}
if ($?) {
&errorMessage(__LINE__);
}
next;
}
else {
$inputfile =~ s/^.+[\\\/]//;
print(STDERR "Concatenating $inputfile...\n");
my @seqfiles;
if ($folder) {
while (my $tempfile = glob("$output/$inputfile.*")) {
if (-z $tempfile) {
unlink($tempfile);
}
else {
push(@seqfiles, $tempfile);
}
}
}
else {
while (my $tempfile = glob("$output.*")) {
if (-z $tempfile) {
unlink($tempfile);
}
else {
push(@seqfiles, $tempfile);
}
}
}
if (@seqfiles) {
if ($folder) {
$filehandleoutput1 = writeFile("$output/$inputfile");
}
else {
$filehandleoutput1 = writeFile("$output");
}
foreach my $seqfile (@seqfiles) {
unless (open($filehandleinput1, "< $seqfile")) {
&errorMessage(__LINE__, "Cannot open \"$seqfile\".");
}
while (<$filehandleinput1>) {
print($filehandleoutput1 $_);
}
close($filehandleinput1);
unlink($seqfile);
}
close($filehandleoutput1);
}
exit;
}
}
# join
while (wait != -1) {
if ($?) {
&errorMessage(__LINE__, 'Cannot concatenate sequence file correctly.');
}
}
}
print(STDERR "done.\n\n");
}
sub processOneSequence {
my ($seqname, $nucseq, $qualseq) = @_;
if ($taxfile && (!defined($otu2newotu{$seqname}) || !defined($newotu2otu{$otu2newotu{$seqname}}) || $newotu2otu{$otu2newotu{$seqname}} ne $seqname) || %otulist && !$otulist{$seqname} || %notulist && $notulist{$seqname} || $minnseq && $members{$seqname} < $minnseq) {
if ($format eq 'FASTQ') {
return('', '');
}
else {
return('');
}
}
if (%keywords || %ngwords) {
my $out = 1;
foreach my $keyword (keys(%keywords)) {
if ($seqname !~ /$keyword/i) {
$out = 0;
}
}
foreach my $ngword (keys(%ngwords)) {
if ($seqname =~ /$ngword/i) {