PGAP.pl

#!/usr/bin/perl

=head1 Name

	PGAP.pl -- A pipeline to annotation prokaryotic genome

=head1 Description

	This is a pipeline to annotation the prokaryote genome. There are six steps:
	Step 1: Gene Denovo Prediction based on HMM, contains Glimmer3 and GeneMarkS two software, default we use 
	Glimmer3 to predict Genes because of its higher accuracy. But GeneMarkS can get more accurat start codon 
	site.
	Step 2: Repeat Finding, contains Tandem Repeat(TR) found by TRF and Transposon element(TE) found by RepeatMasker, by running              RepeatMaskerm, we use the protein lib suppled by RepeatMasker and Repbase database supplied by EMBL. 
	Step 3: ncRNA Finding, contains rRNA predicted by homology blast or RNAmmer, tRNA predicted by tRNAscan, sRNA, snRNA and miRNA            predicted by Rafam.
	Step 4: Gene Function Annotation, we blast the predicted genes with protein database and get the relative function and 
	classification information. The database contains NR, SwissProt, TrEMBL, COG, KEGG.
	Step 5: Draw synteny map, We sort the Scaffolds based on the reference and blast the proteins with reference proteins, then 
	we draw the synteny map based on the sorted Scaffold list and blast result.
	Step 6: Gene Family Analysis, we use Treefam to get gene families and the evolution trees.

=head1 Version
	
	Version: 1.0
	Contact: aipeng@genomics.org.cn

=head1 Usage
	
	perl PGAP.pl [options] Genome.fa
	--all              run all annotation program
	
	--stat             stat the genome scaffolds seqence
	--len <int>        get the scaffolds longer than this len, default 500
	
	--predict <str>    run Gene Prediction, Glimmer3 or GeneMarkS or all, --all default Glimmer3
	--shape <str>      set the shape of prokaryote DNA, circular,linear,partial, default=partial
	
	--repeat
	  --repbase        run RepeatMasker based on Repbase database
	  --proteinmask    run RepeatProteinMasker
	  --trf            run TRF

	--ncRNA
	  --rRNA <str>     search rRNA by RNAmmer or homology or all
	  --rRNA_ref<file> set the reference rRNA seqeunce file, needed for -all
	  --tRNA           search tRNA by tRNAscan
	  --miRNA          search miRNA by blasting with Rfam database
	  --sRNA           search sRNA by blasting with Rfam database
	  --snRNA          search snRNA by blasting with Rfam database
	
	--function <str>   Gene Function Annotation, --all  default:nr-swissprot-trembl-cog-kegg        

	--reference <file> set the references list file  for synteny analysis and Gene family, needed for -all
	--synteny          synteny analysis

	--family           run Treefam to get Gene Family
	--lib <file>       set the lib for Gene Family, default generated by Genome seqence and reference
	--species <file>   species tree, default, created by lib information
	--category <file>  category file, default, created by lib information

	--run <str>        set the parallel type, qsub, or multi, default=qsub
	--cpu <int>        set the cpu number to use in parallel, default 5 for qsub and 20 for multi
	--outdir <str>     set the result directory, default="."
	--prefix <str>     set a prefix name for results
	--help             output help information to screen

=head1 Example

	perl ../PGAP.pl  -all -prefix Ainu711 -reference input/ref.lis -rRNA_ref  input/rRNA_ref.fa -outdir . -cpu 5 -run qsub  input/Ainu711.seq.1000 

=cut

use strict;
use warnings;

use FindBin qw($Bin $Script);
use File::Basename qw(basename dirname);
use Getopt::Long;
use Data::Dumper;
use Pod::Text;

##initialize some parameters fot GetOptions
my $All;
my($Stat,$Length);
my($Predict,$Shape);
my($Repeat,$Repbase,$ProteinMask,$TRF);
my($NcRNA,$RRNA,$RRNA_ref,$TRNA,$MiRNA,$SRNA,$SnRNA);
my($Function);
my($Reference,$Synteny);
my($Family,$Lib,$Species,$Category);
my($Run,$Cpu,$Outdir,$Prefix);
my($Verbose,$Help);

GetOptions(
	"all"=>\$All,
	"stat"=>\$Stat,
	"len:i"=>\$Length,
	"predict:s"=>\$Predict,
	"shape:s"=>\$Shape,
	"repeat"=>\$Repeat,
	"repbase"=>\$Repbase,
	"proteinmask"=>\$ProteinMask,
	"trf"=>\$TRF,
	"ncRNA"=>\$NcRNA,
	"rRNA:s"=>\$RRNA,
    "rRNA_ref:s"=>\$RRNA_ref,
	"tRNA"=>\$TRNA,
	"miRNA"=>\$MiRNA,
	"sRNA"=>\$SRNA,
	"snRNA"=>\$SnRNA,
	"function:s"=>\$Function,
	"reference:s"=>\$Reference,
	"synteny"=>\$Synteny,
	"family"=>\$Family,
	"lib:s"=>\$Lib,
	"species"=>\$Species,
	"category"=>\$Category,
	"run:s"=>\$Run,
	"cpu:i"=>\$Cpu,
	"outdir:s"=>\$Outdir,
	"prefix:s"=>\$Prefix,
	"verbose"=>\$Verbose,
	"help"=>\$Help
);

##get the introduction information
die `pod2text $0` if ($Help || @ARGV==0);

#===============================================================
##set default value for some parameter
$Length ||= 500;
$Shape ||= "partial";
$Run ||= "qsub";
$Cpu ||= $Run eq 'qsub' ? 20 : 5;
$Outdir ||= ".";
$Outdir =~ s/\/$//;

my$seq_file=shift;
my$pwd=$ENV{'PWD'};
$seq_file="$pwd/$seq_file" if($seq_file!~/^\//);
$Outdir="$pwd/$Outdir" if($Outdir!~/^\//);

#$RRNA_ref needed full path
if(defined $RRNA_ref && $RRNA_ref!~/^\//){
	$RRNA_ref="$pwd/$RRNA_ref";
}

my$seq_file_name=basename($seq_file);
$Prefix ||= $seq_file_name;

##defined the work directory and $command
my$cwd=$Outdir;
my$command="";
mkdir $cwd unless (-d $cwd);

$Script="$Bin/script";
mkdir "$Outdir/Result" unless -d "$Outdir/Result";

#================================================================
#make all analysis if defined all
if(defined $All){
	$Stat="";
	$Predict="glimmer";
	$Repeat=$Repbase=$ProteinMask=$TRF="";
	$NcRNA=$TRNA=$MiRNA=$SRNA=$SnRNA="";
	$Function="nr-swissprot-trembl-cog-kegg";
	$Synteny="";
	$Family="";
	if(! defined $Reference){
		die "ERROR:You must defined refernce for -all!\n";
	}
	if(! defined $RRNA_ref){
		$RRNA="RNAmmer";
	}else{
		$RRNA="homology";
	}
}



#========================================================
#        +------------------------+ 
#        |  Genome Scaffold Stat  |
#        |      00.Seqence        |
#        +------------------------+

if(defined $Stat){
	print "cut the Scaffolds ...";
	##creat dir
	$cwd="$Outdir/00.Seqence";
	mkdir $cwd unless (-d $cwd);
	mkdir "$Outdir/Result/00.Seqence" unless -d "$Outdir/Result/00.Seqence";
	
	##generate Scaffolds longer than $Len
	`perl $Script/sequence.pl -l $Length -scaff $cwd/$seq_file_name.$Length  $seq_file`;
	#`$Script/cs $cwd/$seq_file_name.$Length > $cwd/$seq_file_name.$Length.stat`;
	
	##save needed files
	`cp $cwd/$seq_file_name.$Length $Outdir/Result/00.Seqence/$Prefix.seq`;
	#`cp $cwd/$seq_file_name.$Length.stat $Outdir/Result/00.Seqence/$Prefix.seq.stat`;
	my$pwd=$ENV{'PWD'};
	$seq_file="$pwd/00.Seqence/$seq_file_name.$Length";
	$seq_file_name=basename($seq_file);
	print "Done\n";
}

#===========================================================
#          +----------------------+
#          |   Reference files    |
#          | 00.Seqence/reference |
#          +----------------------+

my@Reference;
if(defined $Reference){
	print "Pretreat Reference files ...";
	open(IN,$Reference) || die "$Reference $!\n";
	while(<IN>){
		chomp;
		next if (/^#/);
		push(@Reference , $_);
	}

	##creat dir
	$cwd="$Outdir/00.Seqence/reference";
	mkdir $cwd unless (-d $cwd);
	chdir $cwd;
	
	##get information from Reference list file
	open OUT,">all.lib" || die "all.lib $!\n";
	my$libline="";
	$pwd=$ENV{'PWD'};
	foreach my$ref(@Reference){
		die "Check: $ref is not exists!" if(! -e $ref);
		my$ref_name=basename($ref);
		`cp $ref $ref_name`;
		$libline.="Name=$ref_name\nCds=$pwd/00.Seqence/reference/$ref_name.cds\nPep=$pwd/00.Seqence/reference/$ref_name.pep\n";
		next if(-e "$ref_name.pep" && -e "$ref_name.cds" && -e "$ref_name.seq");
		my$filetype=get_file_type($ref_name);
		if ($filetype eq "GenBank"){
			`perl $Bin/script/genbank_parser.pl $ref_name`
		}elsif($filetype eq "FASTA"){
			`perl $Bin/gene\-prediction/bin/gene-predict.pl --glimmer --prefix $ref_name -shape circular -cpu $Cpu -run $Run -outdir ./ $ref_name`;
			`mv $ref_name.glimmer.pep $ref_name.pep`;
			`mv $ref_name.glimmer.cds $ref_name.cds`;
			`mv $ref_name $ref_name.seq`;
		}	
	}
	print OUT $libline;
	close OUT;
	print "Done\n";
}


#========================================================
#        +------------------------+
#        |    Gene Prediction     |
#        |   01.Gene-Prediction   |
#        +------------------------+

if(defined $Predict){
	print "Gene Prediction ...";
	##creat dir
	$cwd="$Outdir/01.Gene-Prediction";
	mkdir $cwd unless (-d $cwd);
	mkdir "$Outdir/Result/01.Gene-Prediction" unless -d "$Outdir/Result/01.Gene-Prediction";
	chdir $cwd;
	#creat gene predict command and save needed files to Result folder
	if($Predict =~ /all/i ){
		`cp $Bin/gene\-prediction/bin/.gm_key ~/`;
		$command="perl $Bin/gene\-prediction/bin/gene-predict.pl --glimmer --genemark --prefix $Prefix -shape $Shape -cpu $Cpu -run $Run -outdir ./ $seq_file\n";
		$command.="cp $seq_file_name.glimmer.gff ../Result/01.Gene-Prediction/$Prefix.glimmer.gff; cp $seq_file_name.glimmer.cds ../Result/01.Gene-Prediction/$Prefix.glimmer.cds; cp $seq_file_name.glimmer.pep ../Result/01.Gene-Prediction/$Prefix.glimmer.pep\n";
		$command.="cp $seq_file_name.gmhmmp.gff ../Result/01.Gene-Prediction/$Prefix.gmhmmp.gff; cp $seq_file_name.gmhmmp.cds ../Result/01.Gene-Prediction/$Prefix.gmhmmp.cds; cp $seq_file_name.gmhmmp.pep ../Result/01.Gene-Prediction/$Prefix.gmhmmp.pep\n";
	}elsif($Predict =-~ /glimmer/i){
		$command="$Bin/gene\-prediction/bin/gene-predict.pl --glimmer --prefix $Prefix -shape $Shape -cpu $Cpu -run $Run -outdir ./ $seq_file\n";
		$command.="cp $seq_file_name.glimmer.gff ../Result/01.Gene-Prediction/$Prefix.glimmer.gff; cp $seq_file_name.glimmer.cds ../Result/01.Gene-Prediction/$Prefix.glimmer.cds; cp $seq_file_name.glimmer.pep ../Result/01.Gene-Prediction/$Prefix.glimmer.pep\n";
	}elsif($Predict =~ /genemark/i){
		`cp $Bin/gene\-prediction/bin/.gm_key ~/`;
		$command="$Bin/gene\-prediction/bin/gene-predict.pl --genemark --prefix $Prefix -shape $Shape -cpu $Cpu -run $Run -outdir ./ $seq_file\n";
		$command.="cp $seq_file_name.gmhmmp.gff ../Result/01.Gene-Predict/$Prefix.gmhmmp.gff; cp $seq_file_name.gmhmmp.cds ../Result/01.Gene-Prediction/$Prefix.gmhmmp.cds; cp $seq_file_name.gmhmmp.pep ../Result/01.Gene-Prediction/$Prefix.gmhmmp.pep\n";
	}

	runcmd($command,"gene_predict.sh");	

	chdir "../";	

	#stat the gene predict result
	`perl $Bin/script/plot_length.pl Result/01.Gene-Prediction/$Prefix.gmhmmp.cds`	if($Predict =~ /all/i or $Predict =~ /genemark/i);
	`perl $Bin/script/plot_length.pl Result/01.Gene-Prediction/$Prefix.glimmer.cds` if($Predict =~ /all/i or $Predict =~ /glimmer/i );

	print "Done\n";
}

#===================================================================
#           +-------------------------+
#           |    Repeat Finding       |
#           |   02.Repeat-finding     |
#           +-------------------------+


if(defined $Repeat){
	print "Repeat Finding ...";
	##creat dir
	$cwd="$Outdir/02.Repeat-finding";
	mkdir $cwd unless (-d $cwd);
	mkdir "$Outdir/Result/02.Repeat-finding" unless -d "$Outdir/Result/02.Repeat-finding";
	chdir $cwd;

	##creat repeat finding command and save needed files to Result folder
	$command="perl $Bin/repeat/bin/repeat_pipeline.pl ";
	#if(defined $Denovo){
	#	$command.="--denovo --repeatmodeler ";
	#}
	if(defined $Repbase){
		$command.="--repbase ";
	}
	if(defined $ProteinMask){
		$command.="--proteinmasker ";
	}
	if(defined $TRF){
		$command.="--trf ";
	}
	$command.="--outdir ./ --prefix $Prefix --cutf 20 --run $Run --cpu $Cpu $seq_file \n";
	
	#if(defined $Denovo){
	#	$command.="cp 00.Denovo/RepeatModeler/$seq_file_name.RepeatMasker.out ../Result/02.Repeat-finding/$Prefix.RepeatModeler.out\n";
	#	$command.="cp 00.Denovo/RepeatModeler/$seq_file_name.RepeatMasker.out.gff ../Result/02.Repeat-finding/$Prefix.RepeatModeler.out.gff\n";
	#}
	if(defined $Repbase){
		$command.="cp 01.Repbase/$seq_file_name.RepeatMasker.out ../Result/02.Repeat-finding/$Prefix.Repbase.out\n";
		$command.="cp 01.Repbase/$seq_file_name.RepeatMasker.out.gff ../Result/02.Repeat-finding/$Prefix.Repbase.out.gff\n";
	}
	if(defined $ProteinMask){
		$command.="cp 02.RepeatProteinMasker/$seq_file_name.Proteinmask.annot ../Result/02.Repeat-finding/$Prefix.Proteinmask.annot\n";
		$command.="cp 02.RepeatProteinMasker/$seq_file_name.Proteinmask.annot.gff ../Result/02.Repeat-finding/$Prefix.Proteinmask.annot.gff\n";
	}
	if(defined $TRF){
		$command.="cp 03.trf/$seq_file_name.trf.dat ../Result/02.Repeat-finding/$Prefix.trf.dat\n";
		$command.="cp 03.trf/$seq_file_name.trf.dat.gff ../Result/02.Repeat-finding/$Prefix.trf.dat.gff\n";
	}

	##creat and rum repeat finding shell
	runcmd($command,"repeat_finding.sh");

	##stat
	
	chdir "../";

	print "Done\n";

}

#===========================================================================================
#             +--------------------------+
#             |      ncRNA Finding       |
#             |     03.ncRNA-finding     |
#             +--------------------------+#

if(defined $NcRNA){
	print "ncRNA Finding ...";
	##creat dir
	$cwd="$Outdir/03.ncRNA-finding";
	mkdir $cwd unless (-d $cwd);
	mkdir "$Outdir/Result/03.ncRNA-finding" unless -d "$Outdir/Result/03.ncRNA-finding";
	chdir $cwd;

	##creat run ncRNA command
	$command="perl $Bin/ncRNA/bin/find-RNA.pl ";
	if(defined $RRNA){
		$command.="--rRNA $RRNA --ref_rRNA $RRNA_ref " if(defined $RRNA_ref);
		$command.="--rRNA $RRNA " if(! defined $RRNA_ref);
	}
	if(defined $TRNA){
		$command.="--tRNA ";
	}
	if(defined $MiRNA){
		$command.="--MiRNA ";
	}
	if(defined $SRNA){
		$command.="--sRNA ";
	}
	if(defined $SnRNA){
		$command.="--snRNA ";
	}
	$command.="--spec_tag B --species bac --prefix $Prefix --cpu $Cpu --run $Run --outdir ./ $seq_file\n";

	##save needed files to Result folder

	if(defined $MiRNA){
		$command.="cp miRNA/$seq_file_name.miRNA.cmsearch.all.gff ../Result/03.ncRNA-finding/$Prefix.miRNA.cmsearch.all.gff\n";
		$command.="cp miRNA/$seq_file_name.miRNA.cmsearch.confident.gff ../Result/03.ncRNA-finding/$Prefix.miRNA.cmsearch.confident.gff\n";
		$command.="cp miRNA/$seq_file_name.miRNA.cmsearch.confident.nr.gff ../Result/03.ncRNA-finding/$Prefix.miRNA.cmsearch.confident.nr.gff\n";
	}
	if(defined $SnRNA){
		$command.="cp snRNA/$seq_file_name.snRNA.cmsearch.all.gff ../Result/03.ncRNA-finding/$Prefix.snRNA.cmsearch.all.gff\n";
		$command.="cp snRNA/$seq_file_name.snRNA.cmsearch.confident.gff ../Result/03.ncRNA-finding/$Prefix.snRNA.cmsearch.confident.gff\n";
		$command.="cp snRNA/$seq_file_name.snRNA.cmsearch.confident.nr.gff ../Result/03.ncRNA-finding/$Prefix.snRNA.cmsearch.confident.nr.gff\n";
	}
	if(defined $SRNA){
		$command.="cp sRNA/$seq_file_name.sRNA.cmsearch.all.gff ../Result/03.ncRNA-finding/$Prefix.sRNA.cmsearch.all.gff\n";
		$command.="cp sRNA/$seq_file_name.sRNA.cmsearch.confident.gff ../Result/03.ncRNA-finding/$Prefix.sRNA.cmsearch.confident.gff\n";
		$command.="cp sRNA/$seq_file_name.sRNA.cmsearch.confident.nr.gff ../Result/03.ncRNA-finding/$Prefix.sRNA.cmsearch.confident.nr.gff\n";
	}
	if(defined $TRNA){
		$command.="cp tRNA/$seq_file_name.tRNA ../Result/03.ncRNA-finding/$Prefix.tRNA\n";
		$command.="cp tRNA/$seq_file_name.tRNA.gff ../Result/03.ncRNA-finding/$Prefix.tRNA.gff\n";
		$command.="cp tRNA/$seq_file_name.tRNA.structure ../Result/03.ncRNA-finding/$Prefix.tRNA.structure\n";
	}
	if(defined $RRNA_ref){
		$command.="cp rRNA/$seq_file_name.rRNA.blast.tab ../Result/03.ncRNA-finding/$Prefix.rRNA.tab\n";
		$command.="cp rRNA/$seq_file_name.rRNA.blast.tab.gff ../Result/03.ncRNA-finding/$Prefix.rRNA.gff\n";
	}
	if(defined $RRNA && ($RRNA =~ /all/i || $RRNA =~ /RNAmmer/i)){
		$command.="cp rRNA/Denovo/$Prefix.rRNA.fq ../Result/03.ncRNA-finding/$Prefix.rRNA.fq\n";
		$command.="cp rRNA/Denovo/$Prefix.rRNA.gff ../Result/03.ncRNA-finding/$Prefix.rRNA.gff\n";
	}
	#stat
	#
	
	##creat and run ncRNA sell
	runcmd($command,"ncRNA.sh");
	
	chdir "../";

	print "Done\n"
}

#========================================================================
#              +----------------------------+
#              |  Gene Function Annotation  |
#              |     04.gene-function       |
#              +----------------------------+         

if(defined $Function){
	print "Gene Function Annotation ...";
	##creat dir
	$cwd="$Outdir/04.Gene-Function";
	mkdir $cwd unless -d $cwd;
	mkdir "$Outdir/Result/04.Gene-Function" unless -d "$Outdir/Result/04.Gene-Function";
	chdir $cwd;
	
	if(! -e "../01.Gene-Prediction/$seq_file_name.glimmer.pep"){
		die "You must predict genes first!\nIf you want to annotation only, you can turn to $Bin/gene-function/bin/run_annoflow.pl \n";
	}
	##creat gene function annotation command
	$command="perl $Bin/gene-function/bin/run_annoflow.pl -i ../01.Gene-Prediction/$seq_file_name.glimmer.pep -c $Cpu -r $Run -b Y -d 0 -e 1e-5 -m 0 -s $Function \n";

	##creat and run gene function annotation shell
	runcmd($command,"gene_function.sh");

	##save needed files to Result
	$_=$Function;
	if(/nr/){
		`cp AnnoPack/03.anno.nr/nr.list.filter ../Result/04.Gene-Function/$Prefix.nr.list.filter`;
		`cp AnnoPack/03.anno.nr/nr.list.filter.anno ../Result/04.Gene-Function/$Prefix.nr.list.anno`;
	}
	if(/swissprot/){
		`cp AnnoPack/03.anno.swissprot/swissprot.list.filter ../Result/04.Gene-Function/$Prefix.swissprot.list.filter`;
		`cp AnnoPack/03.anno.swissprot/swissprot.list.anno ../Result/04.Gene-Function/$Prefix.swissprot.list.anno`;
	}
	if(/trembl/){
		`cp AnnoPack/03.anno.trembl/trembl.list.filter ../Result/04.Gene-Function/$Prefix.trembl.list.filter`;
		`cp AnnoPack/03.anno.trembl/trembl.list.filter.anno ../Result/04.Gene-Function/$Prefix.trembl.list.anno`;
	}
	if(/cog/){
		`cp AnnoPack/03.anno.cog/cog.list.filter ../Result/04.Gene-Function/$Prefix.cog.list.filter`;
		`cp AnnoPack/03.anno.cog/cog.list.filter.anno ../Result/04.Gene-Function/$Prefix.cog.list.anno`;
		`cp AnnoPack/03.anno.cog/cog.list.cogclass.png ../Result/04.Gene-Function/$Prefix.cog.list.cogclass.png`;
		`cp AnnoPack/03.anno.cog/cog.list.class.catalog ../Result/04.Gene-Function/`;
	}
	if(/kegg/){
		`cp AnnoPack/03.anno.kegg/kegg.list.filter ../Result/04.Gene-Function/$Prefix.kegg.list.filter`;
		`cp AnnoPack/03.anno.kegg/kegg.list.anno ../Result/04.Gene-Function/$Prefix.kegg.list.anno`;
		`cp -r AnnoPack/03.anno.kegg/kegg.list.catalog.map.fig/ ../Result/04.Gene-Function/KEGG_MAP`;
		`cp AnnoPack/03.anno.kegg/functional_classification_2.png ../Result/04.Gene-Function/$Prefix.kegg.functional_classification_2.png`;
	}
	`cp AnnoPack/anno.table ../Result/04.Gene-Function/$Prefix.annotation.table`;

	##stat
	#
	#
	
	chdir "../";	

	print "Done\n";
}

#=================================================================================
#              +----------------------------+
#              |     Synteny Analysis       |
#              |       05.synteny           |
#              +----------------------------+

if (defined $Synteny){
	print "Synteny Analysis ...";

	if(! -e "$Outdir/01.Gene-Prediction/$seq_file_name.glimmer.pep"){
		die "You must predict genes first!\n";
	}
	
	if(! defined $Reference){
		die "Synteny analysis need a reference at least !\n";
	}
	
	$cwd="$Outdir/05.Synteny";
	mkdir $cwd unless -d $cwd;
	mkdir "$Outdir/Result/05.Synteny" unless -d "$Outdir/Result/05.Synteny";
	chdir $cwd;

	foreach my$ref (@Reference){
		my$ref_name=basename($ref);
		
		##creat synteny analysis command
		$command="perl $Bin/synteny/bin/synteny.pl -pep1 ../01.Gene-Prediction/$seq_file_name.glimmer.pep -chr1 ../00.Seqence/$seq_file_name -pep2 ../00.Seqence/reference/$ref_name.pep -chr2 ../00.Seqence/reference/$ref_name.seq -outdir $ref_name -scale 1 -block 1000 -prefix $Prefix\n";
		
		##save need files to Result folder
		$command.="cp $ref_name/*.reciprocal.best.list.synteny.svg ../Result/05.Synteny/$Prefix.$ref_name.synteny.svg\n";
		$command.="cp $ref_name/*.reciprocal.best.stat ../Result/05.Synteny/$Prefix.$ref_name.stat\n";
		$command.="cp $ref_name/*.reciprocal.best.list ../Result/05.Synteny/$Prefix.$ref_name.synteny.list\n";
			
		##creat and run  synteny analysis shell
		runcmd($command,"$ref_name.synteny.sh");
	}

	chdir "../";

	print "Done\n";
}


#=======================================================================================
#               +-----------------------+
#               |     Gene Family       |
#               |    06.Gene-Family     |
#               +-----------------------+

if(defined $Family){
	print "Gene Family ...";
	##creat dir
	$cwd="$Outdir/06.Gene-Family";
	mkdir $cwd unless -d $cwd;
	mkdir "$Outdir/Result/06.Gene-Family" unless -