叶绿体基因组基因、外显子、内含子、基因间隔区提取

Bioinformatic_Scripts/extract_sequences_from_gb_files

一、用途

从注释好的gb格式（GenBank Flat File）的叶绿体基因组文件中批量提取基因（包括内含子）、编码基因（不包括内含子，外显子合并）、基因编码区（外显子单独提取）、基因非编码区（即基因外显子间隔区，包括嵌套基因外显子之间区域、以及内含子）、基因间隔区（不包括嵌套基因外显子之间的区域），以及各种自由组合，具体脚本在文件夹extract_sequences_from_gb_files里面。

二、脚本

1、文件夹extract_coding_noncoding，包括组合提取脚本

（1）提取基因和基因间隔区

基因：包括内含子。
基因间隔区：不包括嵌套基因外显子之间的区域，例如trnK-UUU-2-matK、matK-trnK-UUU-1。
1_extract_bed_gene_and_IGS.pl

#!/usr/bin/perl -w
use strict;
use File::Find;
use Data::Dumper;
$|=1;

print "Please type your input directory:";
my $dirname=;
chomp $dirname;

my $pattern=".gb";
my @filenames;
find(\&target,$dirname);
sub target{
    if (/$pattern/){
        push @filenames,"$File::Find::name";
    }
    return;
}

while (@filenames) {
    my $filename_gb=shift @filenames;
    my $filename_base=$filename_gb;
    $filename_base=~ s/(.*).gb/$1/g;

    open(my $in_gb,"<",$filename_gb);
    open(my $out_gb,">","$filename_base\_temp1");
    while (<$in_gb>){
        $_=~ s/\r\n/\n/g;
        if ($_=~ /\),\n/){
            $_=~ s/\),\n/\),/g;
        }elsif($_=~ /,\n/){
            $_=~ s/,\n/,/g;
        }
        print $out_gb $_;
    }
    close $in_gb;
    close $out_gb;

    open(my $in_gbk,"<","$filename_base\_temp1");
    open(my $out_gbk,">","$filename_base\_temp2");
    while (<$in_gbk>){
        $_=~ s/,\s+/,/g;
        print $out_gbk $_;
    }
    close $in_gbk;
    close $out_gbk;


    #generate_bed_file
    my (@row_array,$species_name,$length,$element,@genearray,@output1);
    my $mark=0;
    open (my $in_genbank,"<","$filename_base\_temp2");
    while (<$in_genbank>){
        chomp;
        @row_array=split /\s+/,$_;
        if (/^LOCUS/i){
            $species_name=$row_array[1];
            $length=$row_array[2];
        }elsif(/ {5}gene {12}/){
            if ($row_array[2]=~ /^\d+..\d+$/){
                $row_array[2]="\+\t$row_array[2]\t$row_array[1]";
                $row_array[2]=~ s/\../\t/g;
            }elsif($row_array[2]=~/^complement\((\d+..\d+)\)$/){
                $row_array[2]="-\t$1\t$row_array[1]";
                $row_array[2]=~ s/\../\t/g;
            }elsif($row_array[2]=~ /^join\((\d+..\d+),(\d+..\d+)\)$/) {
                $row_array[2]="+\t$1\t$row_array[1]\t+\t$2\t$row_array[1]";
                $row_array[2]=~ s/\../\t/g;
            }elsif($row_array[2]=~ /^join\((\d+..\d+),(\d+..\d+),(\d+..\d+)\)$/) {
                $row_array[2]="+\t$1\t$row_array[1]\t+\t$2\t$row_array[1]\t+\t$3\t$row_array[1]";
                $row_array[2]=~ s/\../\t/g;
            }elsif($row_array[2]=~ /^join\(complement\((\d+..\d+)\),complement\((\d+..\d+)\)\)$/){
                $row_array[2]="-\t$1\t$row_array[1]\t-\t$2\t$row_array[1]";
                $row_array[2]=~ s/\../\t/g;
            }elsif($row_array[2]=~ /^join\(complement\((\d+..\d+)\),(\d+..\d+)\)$/){
                $row_array[2]="-\t$1\t$row_array[1]\t+\t$2\t$row_array[1]";
                $row_array[2]=~ s/\../\t/g;
            }elsif($row_array[2]=~ /^join\(complement\((\d+..\d+)\),complement\((\d+..\d+)\),complement\((\d+..\d+)\)\)$/){
                $row_array[2]="-\t$1\t$row_array[1]\t-\t$2\t$row_array[1]\t-\t$3\t$row_array[1]";
                $row_array[2]=~ s/\../\t/g;
            }elsif($row_array[2]=~ /^complement\(join\((\d+..\d+),(\d+..\d+)\)\)$/){
                $row_array[2]="-\t$1\t$row_array[1]\t-\t$2\t$row_array[1]";
                $row_array[2]=~ s/\../\t/g;
            }elsif($row_array[2]=~ /^complement\(join\((\d+..\d+),(\d+..\d+),(\d+..\d+)\)\)$/){
                $row_array[2]="-\t$1\t$row_array[1]\t-\t$2\t$row_array[1]\t-\t$3\t$row_array[1]";
                $row_array[2]=~ s/\../\t/g;
            }elsif($row_array[2]=~ /^order\((\d+..\d+),(\d+..\d+)\)$/){
                $row_array[2]="+\t$1\t$row_array[1]\t+\t$2\t$row_array[1]";
                $row_array[2]=~ s/\../\t/g;
            }elsif($row_array[2]=~ /^order\((\d+..\d+),(\d+..\d+),(\d+..\d+)\)$/){
                $row_array[2]="+\t$1\t$row_array[1]\t+\t$2\t$row_array[1]\t+\t$3\t$row_array[1]";
                $row_array[2]=~ s/\../\t/g;
            }elsif($row_array[2]=~ /^order\(complement\((\d+..\d+)\),complement\((\d+..\d+)\)\)$/){
                $row_array[2]="-\t$1\t$row_array[1]\t-\t$2\t$row_array[1]";
                $row_array[2]=~ s/\../\t/g;
            }elsif($row_array[2]=~ /^order\(complement\((\d+..\d+)\),complement\((\d+..\d+)\),complement\((\d+..\d+)\)\)$/){
                $row_array[2]="-\t$1\t$row_array[1]\t-\t$2\t$row_array[1]\t-\t$3\t$row_array[1]";
                $row_array[2]=~ s/\../\t/g;
            }elsif($row_array[2]=~ /^order\(complement\((\d+..\d+)\),(\d+..\d+)\)$/){
                $row_array[2]="-\t$1\t$row_array[1]\t+\t$2\t$row_array[1]";
                $row_array[2]=~ s/\../\t/g;
            }elsif($row_array[2]=~ /^order\(complement\((\d+..\d+)\),(\d+..\d+),(\d+..\d+)\)$/){
                $row_array[2]="-\t$1\t$row_array[1]\t+\t$2\t$row_array[1]\t+\t$3\t$row_array[1]";
                $row_array[2]=~ s/\../\t/g;
            }elsif($row_array[2]=~ /^join\(complement\((\d+..\d+)\),(\d+..\d+),(\d+..\d+)\)$/){
                $row_array[2]="-\t$1\t$row_array[1]\t+\t$2\t$row_array[1]\t+\t$3\t$row_array[1]";
                $row_array[2]=~ s/\../\t/g;
            }elsif($row_array[2]=~ /^join\((\d+..\d+),(\d+..\d+),complement\((\d+..\d+)\)\)$/) {
                $row_array[2]="+\t$1\t$row_array[1]\t+\t$2\t$row_array[1]\t-\t$3\t$row_array[1]";
                $row_array[2]=~ s/\../\t/g;
            }
            $element=$row_array[2];
            $mark=1;
        }elsif(/^\s+\/gene="(.*)"/ and $mark == 1){
            $element=$species_name.":".$1.":".$element;
            push @genearray,$element;
            $element=();
            $mark=0;
        }
    }
    close $in_genbank;
    foreach (@genearray){
        my @array=split /:/,$_;
        push @output1,"$array[0]\t$array[1]\t$array[2]\n";
    }
    @row_array=();
    @genearray=();


    #put_fasta_sequence_in_array
    my $flag=0;
    my @sequence;
    my (@fas1,@fas2);
    open(my $in_genebank,"<","$filename_base\_temp2");
    while (<$in_genebank>){
        if ($_=~ /ORIGIN/){
            $flag=1;
        }
        if ($_=~ /\/\//){
            $flag=2;
        }
        if ($flag==1){
            next if ($_=~ /ORIGIN/);
            push @sequence,$_;
        }
    }
    close $in_genebank;
    foreach (@sequence){
        chomp;
        $_=~ s/\s*//g;
        $_=~ s/\d+//g;
        push @fas1,$_;
    }
    my $fas1=join "",@fas1;
    my (@fasta1,@fasta2);
    push @fasta1,$species_name,$fas1;
    @fasta2=@fasta1;

    unlink "$filename_base\_temp1";
    unlink "$filename_base\_temp2";


    #edit_bed_file
    my (%SP1,%GENE1,%STRAND1,%START1,%END1,%TYPE1,%STRAND2,%START2,%END2,%TYPE2,%STRAND3,%START3,%END3,%TYPE3,@output2);
    my $cnt1=0;
    foreach (@output1) {
        chomp;
        $cnt1++;
        ($SP1{$cnt1},$GENE1{$cnt1},$STRAND1{$cnt1},$START1{$cnt1},$END1{$cnt1},$TYPE1{$cnt1},$STRAND2{$cnt1},$START2{$cnt1},$END2{$cnt1},$TYPE2{$cnt1})=(split /\s+/,$_)[0,1,2,3,4,5,6,7,8,9];
    }
    foreach (1..$cnt1) {
        if (defined $STRAND2{$_} eq "") {
            push @output2,($SP1{$_}."\t".$GENE1{$_}."\t".$STRAND1{$_}."\t".$START1{$_}."\t".$END1{$_}."\t".$TYPE1{$_});
        }elsif (defined $STRAND2{$_} ne "") {
            if (($STRAND1{$_} eq "-") and ($STRAND2{$_} eq "-") and ($START1{$_} < $START2{$_})){
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-2"."\t".$STRAND1{$_}."\t".$START1{$_}."\t".$END1{$_}."\t".$TYPE1{$_});
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-1"."\t".$STRAND2{$_}."\t".$START2{$_}."\t".$END2{$_}."\t".$TYPE2{$_});
            }elsif(($STRAND1{$_} eq "-") and ($STRAND2{$_} eq "-") and ($START1{$_} > $START2{$_})){
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-1"."\t".$STRAND1{$_}."\t".$START1{$_}."\t".$END1{$_}."\t".$TYPE1{$_});
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-2"."\t".$STRAND2{$_}."\t".$START2{$_}."\t".$END2{$_}."\t".$TYPE2{$_});
            }elsif(($STRAND1{$_} eq "-") and ($STRAND2{$_} eq "+") and ($START1{$_} > $START2{$_})){
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-1"."\t".$STRAND1{$_}."\t".$START1{$_}."\t".$END1{$_}."\t".$TYPE1{$_});
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-2"."\t".$STRAND2{$_}."\t".$START2{$_}."\t".$END2{$_}."\t".$TYPE2{$_});
            }elsif(($STRAND1{$_} eq "+") and ($STRAND2{$_} eq "+") and ($START1{$_} < $START2{$_})){
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-1"."\t".$STRAND1{$_}."\t".$START1{$_}."\t".$END1{$_}."\t".$TYPE1{$_});
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-2"."\t".$STRAND2{$_}."\t".$START2{$_}."\t".$END2{$_}."\t".$TYPE2{$_});
            }elsif(($STRAND1{$_} eq "+") and ($STRAND2{$_} eq "+") and ($START1{$_} > $START2{$_}) and ($GENE1{$_} ne "rps12")){
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-2"."\t".$STRAND1{$_}."\t".$START1{$_}."\t".$END1{$_}."\t".$TYPE1{$_});
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-1"."\t".$STRAND2{$_}."\t".$START2{$_}."\t".$END2{$_}."\t".$TYPE2{$_});
            }elsif(($STRAND1{$_} eq "+") and ($STRAND2{$_} eq "+") and ($START1{$_} > $START2{$_}) and ($GENE1{$_} eq "rps12")){
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-1"."\t".$STRAND1{$_}."\t".$START1{$_}."\t".$END1{$_}."\t".$TYPE1{$_});
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-2"."\t".$STRAND2{$_}."\t".$START2{$_}."\t".$END2{$_}."\t".$TYPE2{$_});
            }
        }
    }
    @output1=();


    #sort_bed_file
    my $col=3;
    my (%sort,@output3);
    foreach (@output2){
        my @row=split /\t/,$_;
        $sort{$_}=$row[$col];
    }
    foreach (sort {$sort{$a} <=> $sort{$b}} keys %sort){
        push @output3,"$_\n";
    }
    @output2=();


    #output_bed_file
    open (my $out_bed,">","$filename_base\_bed_gene_IGS.txt");
    foreach (@output3){
        print $out_bed $_;
    }
    close $out_bed;


    #extract_gene
    my (%SP2,%GENE2,%STRAND4,%START4,%END4,%TYPE4,$seq1);
    my $cnt2=0;
    open(my $out_coding,">","$filename_base\_gene.fasta");
    while (@fasta1){
        my $header=shift @fasta1;
        $seq1=shift @fasta1;
    }
    foreach (@output3){
        chomp;
        $cnt2++;
        ($SP2{$cnt2},$GENE2{$cnt2},$STRAND4{$cnt2},$START4{$cnt2},$END4{$cnt2},$TYPE4{$cnt2})=(split /\s+/,$_)[0,1,2,3,4,5];
        my $str=substr($seq1,($START4{$cnt2}-1),($END4{$cnt2}-$START4{$cnt2}+1));
        if ($STRAND4{$cnt2} eq "-") {
            my $rev_com=reverse $str;
            $rev_com=~ tr/ACGTacgt/TGCAtgca/;
            print $out_coding ">".$GENE2{$cnt2}."_".$SP2{$cnt2}."\n".$rev_com."\n";
        }elsif($STRAND4{$cnt2} eq "+"){
            print $out_coding ">".$GENE2{$cnt2}."_".$SP2{$cnt2}."\n".$str."\n";
        }
    }
    close $out_coding;


    #generate_IGS_ranges
    my (%SP3,%GENE3,%STRAND5,%START5,%END5,%TYPE5,$last0,$last1,$last2,@output4);
    my $cnt3=0;
    foreach (@output3){
        chomp;
        $cnt3++;
        ($SP3{$cnt3},$GENE3{$cnt3},$STRAND5{$cnt3},$START5{$cnt3},$END5{$cnt3},$TYPE5{$cnt3})=(split /\s+/,$_)[0,1,2,3,4,5];
    }
    foreach (keys %SP3){
        if ($_==1 and $START5{$_}!=1){
            unshift @output4,$SP3{$_}."\t"."start".'-'.$GENE3{$_}."\t"."?"."/".$STRAND5{$_}."\t"."1"."\t".($START5{$_}-1)."\t"."?"."/".$TYPE5{$_}."\n";
        }
    }
    foreach (1..($cnt3-1)) {
        $last0=$_-1;
        $last1=$_+1;
        $last2=$_+2;
        next if ((($END5{$_}+1) >= ($START5{$last1}-1)) and (($END5{$_}+1) < ($END5{$last1}-1)));
        next if (($_ > 1) and (($END5{$_}+1) < ($END5{$last0}-1)) and (($END5{$_}+1) < ($START5{$last2}-1)));
        if ((($END5{$_}+1) >= ($START5{$last1}-1)) and (($END5{$_}+1) >= ($END5{$last1}-1))){
            push @output4,$SP3{$_}."\t".$GENE3{$_}.'-'.$GENE3{$last2}."\t".$STRAND5{$_}."/".$STRAND5{$last2}."\t".($END5{$_}+1)."\t".($START5{$last2}-1)."\t".$TYPE5{$_}."/".$TYPE5{$last2}."\n";
        }else{
            push @output4,$SP3{$_}."\t".$GENE3{$_}.'-'.$GENE3{$last1}."\t".$STRAND5{$_}."/".$STRAND5{$last1}."\t".($END5{$_}+1)."\t".($START5{$last1}-1)."\t".$TYPE5{$_}."/".$TYPE5{$last1}."\n";
        }
    }
    foreach (keys %SP3){
        if ($_==$cnt3){
            push @output4,$SP3{$_}."\t".$GENE3{$_}.'-'."end"."\t".$STRAND5{$_}."/"."?"."\t".($END5{$_}+1)."\t".$length."\t".$TYPE5{$_}."/"."?"."\n";
        }
    }
    @output3=();


    #extract_IGS
    my (%SP4,%GENE4,%STRAND6,%START6,%END6,%TYPE6,$seq2);
    my $cnt4=0;
    open(my $out_noncoding,">","$filename_base\_IGS.fasta");
    while (@fasta2){
        my $header=shift @fasta2;
        $seq2=shift @fasta2;
    }
    foreach (@output4){
        chomp;
        $cnt4++;
        ($SP4{$cnt4},$GENE4{$cnt4},$STRAND6{$cnt4},$START6{$cnt4},$END6{$cnt4},$TYPE6{$cnt4})=(split /\s+/,$_)[0,1,2,3,4,5];
        my $str=substr($seq2,($START6{$cnt4}-1),($END6{$cnt4}-$START6{$cnt4}+1));
        print $out_noncoding ">".$GENE4{$cnt4}."_".$SP4{$cnt4}."\n".$str."\n";
    }
    @output4=();
    close $out_noncoding;
    #unlink "$filename_base\_IGS.fasta";
}

（2）提取编码基因和基因外显子间隔区

编码基因：不包括内含子，外显子合并。
基因外显子间隔区：包括嵌套基因外显子之间的区域，例如trnK-UUU-2-matK、matK-trnK-UUU-1；包括内含子。
2_extract_bed_CDS_RNA_and_intergenic.pl

#!/usr/bin/perl -w
use strict;
use File::Find;
use Data::Dumper;
$|=1;

print "Please type your input directory:";
my $dirname=;
chomp $dirname;

my $pattern=".gb";
my @filenames;
find(\&target,$dirname);
sub target{
    if (/$pattern/){
        push @filenames,"$File::Find::name";
    }
    return;
}

while (@filenames) {
    my $filename_gb=shift @filenames;
    my $filename_base=$filename_gb;
    $filename_base=~ s/(.*).gb/$1/g;

    open(my $in_gb,"<",$filename_gb);
    open(my $out_gb,">","$filename_base\_temp1");
    while (<$in_gb>){
        $_=~ s/\r\n/\n/g;
        if ($_=~ /\),\n/){
            $_=~ s/\),\n/\),/g;
        }elsif($_=~ /,\n/){
            $_=~ s/,\n/,/g;
        }
        print $out_gb $_;
    }
    close $in_gb;
    close $out_gb;

    open(my $in_gbk,"<","$filename_base\_temp1");
    open(my $out_gbk,">","$filename_base\_temp2");
    while (<$in_gbk>){
        $_=~ s/,\s+/,/g;
        print $out_gbk $_;
    }
    close $in_gbk;
    close $out_gbk;


    #generate_bed_file
    my (@row_array,$species_name,$length,$element,@genearray,@output1);
    my $mark=0;
    open (my $in_genbank,"<","$filename_base\_temp2");
    while (<$in_genbank>){
        chomp;
        @row_array=split /\s+/,$_;
        if (/^LOCUS/i){
            $species_name=$row_array[1];
            $length=$row_array[2];
        }elsif(/ {5}CDS {13}/ or / {5}tRNA {12}/ or / {5}rRNA {12}/){
            if ($row_array[2]=~ /^\d+..\d+$/){
                $row_array[2]="\+\t$row_array[2]\t$row_array[1]";
                $row_array[2]=~ s/\../\t/g;
            }elsif($row_array[2]=~/^complement\((\d+..\d+)\)$/){
                $row_array[2]="-\t$1\t$row_array[1]";
                $row_array[2]=~ s/\../\t/g;
            }elsif($row_array[2]=~ /^join\((\d+..\d+),(\d+..\d+)\)$/) {
                $row_array[2]="+\t$1\t$row_array[1]\t+\t$2\t$row_array[1]";
                $row_array[2]=~ s/\../\t/g;
            }elsif($row_array[2]=~ /^join\((\d+..\d+),(\d+..\d+),(\d+..\d+)\)$/) {
                $row_array[2]="+\t$1\t$row_array[1]\t+\t$2\t$row_array[1]\t+\t$3\t$row_array[1]";
                $row_array[2]=~ s/\../\t/g;
            }elsif($row_array[2]=~ /^join\(complement\((\d+..\d+)\),complement\((\d+..\d+)\)\)$/){
                $row_array[2]="-\t$1\t$row_array[1]\t-\t$2\t$row_array[1]";
                $row_array[2]=~ s/\../\t/g;
            }elsif($row_array[2]=~ /^join\(complement\((\d+..\d+)\),complement\((\d+..\d+)\),complement\((\d+..\d+)\)\)$/){
                $row_array[2]="-\t$1\t$row_array[1]\t-\t$2\t$row_array[1]\t-\t$3\t$row_array[1]";
                $row_array[2]=~ s/\../\t/g;
            }elsif($row_array[2]=~ /^complement\(join\((\d+..\d+),(\d+..\d+)\)\)$/){
                $row_array[2]="-\t$1\t$row_array[1]\t-\t$2\t$row_array[1]";
                $row_array[2]=~ s/\../\t/g;
            }elsif($row_array[2]=~ /^complement\(join\((\d+..\d+),(\d+..\d+),(\d+..\d+)\)\)$/){
                $row_array[2]="-\t$1\t$row_array[1]\t-\t$2\t$row_array[1]\t-\t$3\t$row_array[1]";
                $row_array[2]=~ s/\../\t/g;
            }elsif($row_array[2]=~ /^order\((\d+..\d+),(\d+..\d+)\)$/){
                $row_array[2]="+\t$1\t$row_array[1]\t+\t$2\t$row_array[1]";
                $row_array[2]=~ s/\../\t/g;
            }elsif($row_array[2]=~ /^order\((\d+..\d+),(\d+..\d+),(\d+..\d+)\)$/){
                $row_array[2]="+\t$1\t$row_array[1]\t+\t$2\t$row_array[1]\t+\t$3\t$row_array[1]";
                $row_array[2]=~ s/\../\t/g;
            }elsif($row_array[2]=~ /^order\(complement\((\d+..\d+)\),complement\((\d+..\d+)\)\)$/){
                $row_array[2]="-\t$1\t$row_array[1]\t-\t$2\t$row_array[1]";
                $row_array[2]=~ s/\../\t/g;
            }elsif($row_array[2]=~ /^order\(complement\((\d+..\d+)\),complement\((\d+..\d+)\),complement\((\d+..\d+)\)\)$/){
                $row_array[2]="-\t$1\t$row_array[1]\t-\t$2\t$row_array[1]\t-\t$3\t$row_array[1]";
                $row_array[2]=~ s/\../\t/g;
            }elsif($row_array[2]=~ /^order\(complement\((\d+..\d+)\),(\d+..\d+)\)$/){
                $row_array[2]="-\t$1\t$row_array[1]\t+\t$2\t$row_array[1]";
                $row_array[2]=~ s/\../\t/g;
            }elsif($row_array[2]=~ /^order\(complement\((\d+..\d+)\),(\d+..\d+),(\d+..\d+)\)$/){
                $row_array[2]="-\t$1\t$row_array[1]\t+\t$2\t$row_array[1]\t+\t$3\t$row_array[1]";
                $row_array[2]=~ s/\../\t/g;
            }elsif($row_array[2]=~ /^join\(complement\((\d+..\d+)\),(\d+..\d+),(\d+..\d+)\)$/){
                $row_array[2]="-\t$1\t$row_array[1]\t+\t$2\t$row_array[1]\t+\t$3\t$row_array[1]";
                $row_array[2]=~ s/\../\t/g;
            }elsif($row_array[2]=~ /^join\((\d+..\d+),(\d+..\d+),complement\((\d+..\d+)\)\)$/) {
                $row_array[2]="+\t$1\t$row_array[1]\t+\t$2\t$row_array[1]\t-\t$3\t$row_array[1]";
                $row_array[2]=~ s/\../\t/g;
            }
            $element=$row_array[2];
            $mark=1;
        }elsif(/^\s+\/gene="(.*)"/ and $mark == 1){
            $element=$species_name.":".$1.":".$element;
            push @genearray,$element;
            $element=();
            $mark=0;
        }
    }
    close $in_genbank;
    foreach (@genearray){
        my @array=split /:/,$_;
        push @output1,"$array[0]\t$array[1]\t$array[2]\n";
    }
    @row_array=();
    @genearray=();


    #put_fasta_sequence_in_array
    my $flag=0;
    my @sequence;
    my (@fas1,@fas2);
    open(my $in_genebank,"<","$filename_base\_temp2");
    while (<$in_genebank>){
        if ($_=~ /ORIGIN/){
            $flag=1;
        }
        if ($_=~ /\/\//){
            $flag=2;
        }
        if ($flag==1){
            next if ($_=~ /ORIGIN/);
            push @sequence,$_;
        }
    }
    close $in_genebank;
    foreach (@sequence){
        chomp;
        $_=~ s/\s*//g;
        $_=~ s/\d+//g;
        push @fas1,$_;
    }
    my $fas1=join "",@fas1;
    my (@fasta1,@fasta2);
    push @fasta1,$species_name,$fas1;
    @fasta2=@fasta1;

    unlink "$filename_base\_temp1";
    unlink "$filename_base\_temp2";


    #edit_bed_file
    my (%SP1,%GENE1,%STRAND1,%START1,%END1,%TYPE1,%STRAND2,%START2,%END2,%TYPE2,%STRAND3,%START3,%END3,%TYPE3,@output2);
    my $cnt1=0;
    foreach (@output1) {
        chomp;
        $cnt1++;
        ($SP1{$cnt1},$GENE1{$cnt1},$STRAND1{$cnt1},$START1{$cnt1},$END1{$cnt1},$TYPE1{$cnt1},$STRAND2{$cnt1},$START2{$cnt1},$END2{$cnt1},$TYPE2{$cnt1},$STRAND3{$cnt1},$START3{$cnt1},$END3{$cnt1},$TYPE3{$cnt1})=(split /\s+/,$_)[0,1,2,3,4,5,6,7,8,9,10,11,12,13];
    }

    foreach (1..$cnt1) {
        if (defined $STRAND2{$_} eq "") {
            push @output2,($SP1{$_}."\t".$GENE1{$_}."\t".$STRAND1{$_}."\t".$START1{$_}."\t".$END1{$_}."\t".$TYPE1{$_});
        }elsif ((defined $STRAND2{$_} ne "") and (defined $STRAND3{$_} eq "")) {
            if (($STRAND1{$_} eq "-") and ($START1{$_} < $START2{$_})){
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-2"."\t".$STRAND1{$_}."\t".$START1{$_}."\t".$END1{$_}."\t".$TYPE1{$_});
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-1"."\t".$STRAND2{$_}."\t".$START2{$_}."\t".$END2{$_}."\t".$TYPE2{$_});
            }elsif(($STRAND1{$_} eq "-") and ($START1{$_} > $START2{$_})){
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-1"."\t".$STRAND1{$_}."\t".$START1{$_}."\t".$END1{$_}."\t".$TYPE1{$_});
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-2"."\t".$STRAND2{$_}."\t".$START2{$_}."\t".$END2{$_}."\t".$TYPE2{$_});
            }elsif(($STRAND1{$_} eq "+") and ($START1{$_} < $START2{$_})){
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-1"."\t".$STRAND1{$_}."\t".$START1{$_}."\t".$END1{$_}."\t".$TYPE1{$_});
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-2"."\t".$STRAND2{$_}."\t".$START2{$_}."\t".$END2{$_}."\t".$TYPE2{$_});
            }elsif(($STRAND1{$_} eq "+") and ($START1{$_} > $START2{$_})){
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-2"."\t".$STRAND1{$_}."\t".$START1{$_}."\t".$END1{$_}."\t".$TYPE1{$_});
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-1"."\t".$STRAND2{$_}."\t".$START2{$_}."\t".$END2{$_}."\t".$TYPE2{$_});
            }
        }elsif ((defined $STRAND2{$_} ne "") and (defined $STRAND3{$_} ne "")) {
            if (($STRAND1{$_} eq "-") and ($START1{$_} < $START2{$_}) and ($START2{$_} < $START3{$_})){
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-3"."\t".$STRAND1{$_}."\t".$START1{$_}."\t".$END1{$_}."\t".$TYPE1{$_});
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-2"."\t".$STRAND2{$_}."\t".$START2{$_}."\t".$END2{$_}."\t".$TYPE2{$_});
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-1"."\t".$STRAND3{$_}."\t".$START3{$_}."\t".$END3{$_}."\t".$TYPE3{$_});
            }elsif(($STRAND1{$_} eq "-") and ($START1{$_} > $START2{$_}) and ($START2{$_} > $START3{$_})){
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-1"."\t".$STRAND1{$_}."\t".$START1{$_}."\t".$END1{$_}."\t".$TYPE1{$_});
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-2"."\t".$STRAND2{$_}."\t".$START2{$_}."\t".$END2{$_}."\t".$TYPE2{$_});
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-3"."\t".$STRAND3{$_}."\t".$START3{$_}."\t".$END3{$_}."\t".$TYPE3{$_});
            }elsif(($STRAND1{$_} eq "-") and ($START1{$_} > $START3{$_}) and ($START3{$_} > $START2{$_})){
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-1"."\t".$STRAND1{$_}."\t".$START1{$_}."\t".$END1{$_}."\t".$TYPE1{$_});
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-2"."\t".$STRAND2{$_}."\t".$START2{$_}."\t".$END2{$_}."\t".$TYPE2{$_});
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-3"."\t".$STRAND3{$_}."\t".$START3{$_}."\t".$END3{$_}."\t".$TYPE3{$_});
            }elsif(($STRAND1{$_} eq "+") and ($START1{$_} < $START2{$_}) and ($START2{$_} < $START3{$_})){
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-1"."\t".$STRAND1{$_}."\t".$START1{$_}."\t".$END1{$_}."\t".$TYPE1{$_});
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-2"."\t".$STRAND2{$_}."\t".$START2{$_}."\t".$END2{$_}."\t".$TYPE2{$_});
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-3"."\t".$STRAND3{$_}."\t".$START3{$_}."\t".$END3{$_}."\t".$TYPE3{$_});
            }elsif(($STRAND1{$_} eq "+") and ($START1{$_} > $START2{$_}) and ($START2{$_} > $START3{$_})){
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-3"."\t".$STRAND1{$_}."\t".$START1{$_}."\t".$END1{$_}."\t".$TYPE1{$_});
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-2"."\t".$STRAND2{$_}."\t".$START2{$_}."\t".$END2{$_}."\t".$TYPE2{$_});
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-1"."\t".$STRAND3{$_}."\t".$START3{$_}."\t".$END3{$_}."\t".$TYPE3{$_});
            }elsif(($STRAND1{$_} eq "+") and ($START1{$_} > $START3{$_}) and ($START3{$_} > $START2{$_})){
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-1"."\t".$STRAND1{$_}."\t".$START1{$_}."\t".$END1{$_}."\t".$TYPE1{$_});
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-2"."\t".$STRAND2{$_}."\t".$START2{$_}."\t".$END2{$_}."\t".$TYPE2{$_});
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-3"."\t".$STRAND3{$_}."\t".$START3{$_}."\t".$END3{$_}."\t".$TYPE3{$_});
            }
        }
    }


    #sort_bed_file
    my $col=3;
    my (%sort,@output3);
    foreach (@output2){
        my @row=split /\t/,$_;
        $sort{$_}=$row[$col];
    }
    foreach (sort {$sort{$a} <=> $sort{$b}} keys %sort){
        push @output3,"$_\n";
    }
    @output2=();


    #output_bed_file
    open (my $out_bed,">","$filename_base\_bed_CDS_RNA_intergenic.txt");
    foreach (@output3){
        print $out_bed $_;
    }
    close $out_bed;


    #extract_gene
    my (%SP2,%GENE2,%STRAND4,%START4,%END4,%TYPE4,%STRAND5,%START5,%END5,%TYPE5,%STRAND6,%START6,%END6,%TYPE6,$seq1);
    my $cnt2=0;
    open(my $out_coding,">","$filename_base\_CDS_RNA.fasta");
    while (@fasta1){
        my $header=shift @fasta1;
        $seq1=shift @fasta1;
    }
    foreach (@output1){
        chomp;
        $cnt2++;
        ($SP2{$cnt2},$GENE2{$cnt2},$STRAND4{$cnt2},$START4{$cnt2},$END4{$cnt2},$TYPE4{$cnt2},$STRAND5{$cnt2},$START5{$cnt2},$END5{$cnt2},$TYPE5{$cnt2},$STRAND6{$cnt2},$START6{$cnt2},$END6{$cnt2},$TYPE6{$cnt2})=(split /\s+/,$_)[0,1,2,3,4,5,6,7,8,9,10,11,12,13];
        if (defined $STRAND5{$cnt2} eq "") {
            my $str1=substr($seq1,($START4{$cnt2}-1),($END4{$cnt2}-$START4{$cnt2}+1));
            if ($STRAND4{$cnt2} eq "-") {
                my $rev_com1=reverse $str1;
                $rev_com1=~ tr/ACGTacgt/TGCAtgca/;
                print $out_coding ">".$GENE2{$cnt2}."_".$SP2{$cnt2}."\n".$rev_com1."\n";
            }elsif($STRAND4{$cnt2} eq "+"){
                print $out_coding ">".$GENE2{$cnt2}."_".$SP2{$cnt2}."\n".$str1."\n";
            }
        }elsif((defined $STRAND5{$cnt2} ne "") and (defined $STRAND6{$cnt2} eq "")) {
            if (($STRAND4{$cnt2} eq "-") and ($START4{$cnt2} < $START5{$cnt2})) {
                my $str2=substr($seq1,($START4{$cnt2}-1),($END4{$cnt2}-$START4{$cnt2}+1)).substr($seq1,($START5{$cnt2}-1),($END5{$cnt2}-$START5{$cnt2}+1));
                my $rev_com2=reverse $str2;
                $rev_com2=~ tr/ACGTacgt/TGCAtgca/;
                print $out_coding ">".$GENE2{$cnt2}."_".$SP2{$cnt2}."\n".$rev_com2."\n";
            }elsif(($STRAND4{$cnt2} eq "-") and ($START4{$cnt2} > $START5{$cnt2})) {
                my $str3=substr($seq1,($START5{$cnt2}-1),($END5{$cnt2}-$START5{$cnt2}+1)).substr($seq1,($START4{$cnt2}-1),($END4{$cnt2}-$START4{$cnt2}+1));
                my $rev_com3=reverse $str3;
                $rev_com3=~ tr/ACGTacgt/TGCAtgca/;
                print $out_coding ">".$GENE2{$cnt2}."_".$SP2{$cnt2}."\n".$rev_com3."\n";
            }elsif(($STRAND4{$cnt2} eq "+") and ($START4{$cnt2} < $START5{$cnt2})){
                my $str4=substr($seq1,($START4{$cnt2}-1),($END4{$cnt2}-$START4{$cnt2}+1)).substr($seq1,($START5{$cnt2}-1),($END5{$cnt2}-$START5{$cnt2}+1));
                print $out_coding ">".$GENE2{$cnt2}."_".$SP2{$cnt2}."\n".$str4."\n";
            }elsif(($STRAND4{$cnt2} eq "+") and ($START4{$cnt2} > $START5{$cnt2})){
                my $str5=substr($seq1,($START5{$cnt2}-1),($END5{$cnt2}-$START5{$cnt2}+1)).substr($seq1,($START4{$cnt2}-1),($END4{$cnt2}-$START4{$cnt2}+1));
                print $out_coding ">".$GENE2{$cnt2}."_".$SP2{$cnt2}."\n".$str5."\n";
            }
        }elsif ((defined $STRAND5{$cnt2} ne "") and (defined $STRAND6{$cnt2} ne "")) {
            if (($STRAND4{$cnt2} eq "-") and ($START4{$cnt2} < $START5{$cnt2}) and ($START5{$cnt2} < $START6{$cnt2})) {
                my $str6=substr($seq1,($START4{$cnt2}-1),($END4{$cnt2}-$START4{$cnt2}+1)).substr($seq1,($START5{$cnt2}-1),($END5{$cnt2}-$START5{$cnt2}+1)).substr($seq1,($START6{$cnt2}-1),($END6{$cnt2}-$START6{$cnt2}+1));
                my $rev_com4=reverse $str6;
                $rev_com4=~ tr/ACGTacgt/TGCAtgca/;
                print $out_coding ">".$GENE2{$cnt2}."_".$SP2{$cnt2}."\n".$rev_com4."\n";
            }elsif(($STRAND4{$cnt2} eq "-") and ($START4{$cnt2} > $START5{$cnt2}) and ($START5{$cnt2} > $START6{$cnt2})) {
                my $str7=substr($seq1,($START6{$cnt2}-1),($END6{$cnt2}-$START6{$cnt2}+1)).substr($seq1,($START5{$cnt2}-1),($END5{$cnt2}-$START5{$cnt2}+1)).substr($seq1,($START4{$cnt2}-1),($END4{$cnt2}-$START4{$cnt2}+1));
                my $rev_com5=reverse $str7;
                $rev_com5=~ tr/ACGTacgt/TGCAtgca/;
                print $out_coding ">".$GENE2{$cnt2}."_".$SP2{$cnt2}."\n".$rev_com5."\n";
            }elsif(($STRAND4{$cnt2} eq "-") and ($START4{$cnt2} > $START6{$cnt2}) and ($START6{$cnt2} > $START5{$cnt2})) {
                my $str8=substr($seq1,($START4{$cnt2}-1),($END4{$cnt2}-$START4{$cnt2}+1));
                my $str9=substr($seq1,($START5{$cnt2}-1),($END5{$cnt2}-$START5{$cnt2}+1)).substr($seq1,($START6{$cnt2}-1),($END6{$cnt2}-$START6{$cnt2}+1));
                my $rev_com6=reverse $str8;
                $rev_com6=~ tr/ACGTacgt/TGCAtgca/;
                print $out_coding ">".$GENE2{$cnt2}."_".$SP2{$cnt2}."\n".$rev_com6.$str9."\n";
            }elsif(($STRAND4{$cnt2} eq "+") and ($START4{$cnt2} < $START5{$cnt2}) and ($START5{$cnt2} < $START6{$cnt2})){
                my $str10=substr($seq1,($START4{$cnt2}-1),($END4{$cnt2}-$START4{$cnt2}+1)).substr($seq1,($START5{$cnt2}-1),($END5{$cnt2}-$START5{$cnt2}+1)).substr($seq1,($START6{$cnt2}-1),($END6{$cnt2}-$START6{$cnt2}+1));
                print $out_coding ">".$GENE2{$cnt2}."_".$SP2{$cnt2}."\n".$str10."\n";
            }elsif(($STRAND4{$cnt2} eq "+") and ($START4{$cnt2} > $START5{$cnt2}) and ($START5{$cnt2} > $START6{$cnt2})){
                my $str11=substr($seq1,($START6{$cnt2}-1),($END6{$cnt2}-$START6{$cnt2}+1)).substr($seq1,($START5{$cnt2}-1),($END5{$cnt2}-$START5{$cnt2}+1)).substr($seq1,($START4{$cnt2}-1),($END4{$cnt2}-$START4{$cnt2}+1));
                print $out_coding ">".$GENE2{$cnt2}."_".$SP2{$cnt2}."\n".$str11."\n";
            }elsif(($STRAND4{$cnt2} eq "+") and ($START4{$cnt2} > $START6{$cnt2}) and ($START6{$cnt2} > $START5{$cnt2})) {
                my $str12=substr($seq1,($START4{$cnt2}-1),($END4{$cnt2}-$START4{$cnt2}+1)).substr($seq1,($START5{$cnt2}-1),($END5{$cnt2}-$START5{$cnt2}+1)).substr($seq1,($START6{$cnt2}-1),($END6{$cnt2}-$START6{$cnt2}+1));
                print $out_coding ">".$GENE2{$cnt2}."_".$SP2{$cnt2}."\n".$str12."\n";
            }
        }
    }
    close $out_coding;
    @output1=();


    #generate_IGS_ranges
    my (%SP3,%GENE3,%STRAND7,%START7,%END7,%TYPE7,$last0,$last1,$last2,@output4);
    my $cnt3=0;
    foreach (@output3){
        chomp;
        $cnt3++;
        ($SP3{$cnt3},$GENE3{$cnt3},$STRAND7{$cnt3},$START7{$cnt3},$END7{$cnt3},$TYPE7{$cnt3})=(split /\s+/,$_)[0,1,2,3,4,5];
    }
    foreach (keys %SP3){
        if ($_==1 and $START7{$_}!=1){
            unshift @output4,$SP3{$_}."\t"."start".'-'.$GENE3{$_}."\t"."?"."/".$STRAND7{$_}."\t"."1"."\t".($START7{$_}-1)."\t"."?"."/".$TYPE7{$_}."\n";
        }
    }
    foreach (1..($cnt3-1)) {
        $last0=$_-1;
        $last1=$_+1;
        $last2=$_+2;
        next if ((($END7{$_}+1) >= ($START7{$last1}-1)) and (($END7{$_}+1) < ($END7{$last1}-1)));
        next if (($_ > 1) and (($END7{$_}+1) < ($END7{$last0}-1)) and (($END7{$_}+1) < ($START7{$last2}-1)));
        if ((($END7{$_}+1) >= ($START7{$last1}-1)) and (($END7{$_}+1) >= ($END7{$last1}-1))){
            push @output4,$SP3{$_}."\t".$GENE3{$_}.'-'.$GENE3{$last2}."\t".$STRAND7{$_}."/".$STRAND7{$last2}."\t".($END7{$_}+1)."\t".($START7{$last2}-1)."\t".$TYPE7{$_}."/".$TYPE7{$last2}."\n";
        }else{
            push @output4,$SP3{$_}."\t".$GENE3{$_}.'-'.$GENE3{$last1}."\t".$STRAND7{$_}."/".$STRAND7{$last1}."\t".($END7{$_}+1)."\t".($START7{$last1}-1)."\t".$TYPE7{$_}."/".$TYPE7{$last1}."\n";
        }
    }
    foreach (keys %SP3){
        if ($_==$cnt3){
            push @output4,$SP3{$_}."\t".$GENE3{$_}.'-'."end"."\t".$STRAND7{$_}."/"."?"."\t".($END7{$_}+1)."\t".$length."\t".$TYPE7{$_}."/"."?"."\n";
        }
    }
    @output3=();


    #extract_IGS
    my (%SP4,%GENE4,%STRAND8,%START8,%END8,%TYPE8,$seq2);
    my $cnt4=0;
    open(my $out_noncoding,">","$filename_base\_intergenic.fasta");
    while (@fasta2){
        my $header=shift @fasta2;
        $seq2=shift @fasta2;
    }
    foreach (@output4){
        chomp;
        $cnt4++;
        ($SP4{$cnt4},$GENE4{$cnt4},$STRAND8{$cnt4},$START8{$cnt4},$END8{$cnt4},$TYPE8{$cnt4})=(split /\s+/,$_)[0,1,2,3,4,5];
        my $str=substr($seq2,($START8{$cnt4}-1),($END8{$cnt4}-$START8{$cnt4}+1));
        print $out_noncoding ">".$GENE4{$cnt4}."_".$SP4{$cnt4}."\n".$str."\n";
    }
    @output4=();
    close $out_noncoding;
    #unlink "$filename_base\_intergenic.fasta";
}

（3）提取基因编码区和基因非编码区

基因编码区：外显子单独提取，例如trnK-UUU-2、trnK-UUU-1。
基因非编码区：包括嵌套基因外显子之间的区域，例如trnK-UUU-2-matK、matK-trnK-UUU-1；包括内含子。同脚本2_extract_bed_CDS_RNA_and_intergenic.pl基因外显子间隔区。
3_extract_bed_coding_and_noncoding.pl

#!/usr/bin/perl -w
use strict;
use File::Find;
use Data::Dumper;
$|=1;

print "Please type your input directory:";
my $dirname=;
chomp $dirname;

my $pattern=".gb";
my @filenames;
find(\&target,$dirname);
sub target{
    if (/$pattern/){
        push @filenames,"$File::Find::name";
    }
    return;
}

while (@filenames) {
    my $filename_gb=shift @filenames;
    my $filename_base=$filename_gb;
    $filename_base=~ s/(.*).gb/$1/g;

    open(my $in_gb,"<",$filename_gb);
    open(my $out_gb,">","$filename_base\_temp1");
    while (<$in_gb>){
        $_=~ s/\r\n/\n/g;
        if ($_=~ /\),\n/){
            $_=~ s/\),\n/\),/g;
        }elsif($_=~ /,\n/){
            $_=~ s/,\n/,/g;
        }
        print $out_gb $_;
    }
    close $in_gb;
    close $out_gb;

    open(my $in_gbk,"<","$filename_base\_temp1");
    open(my $out_gbk,">","$filename_base\_temp2");
    while (<$in_gbk>){
        $_=~ s/,\s+/,/g;
        print $out_gbk $_;
    }
    close $in_gbk;
    close $out_gbk;


    #generate_bed_file
    my (@row_array,$species_name,$length,$element,@genearray,@output1);
    my $mark=0;
    open (my $in_genbank,"<","$filename_base\_temp2");
    while (<$in_genbank>){
        chomp;
        @row_array=split /\s+/,$_;
        if (/^LOCUS/i){
            $species_name=$row_array[1];
            $length=$row_array[2];
        }elsif(/ {5}CDS {13}/ or / {5}tRNA {12}/ or / {5}rRNA {12}/){
            if ($row_array[2]=~ /^\d+..\d+$/){
                $row_array[2]="\+\t$row_array[2]\t$row_array[1]";
                $row_array[2]=~ s/\../\t/g;
            }elsif($row_array[2]=~/^complement\((\d+..\d+)\)$/){
                $row_array[2]="-\t$1\t$row_array[1]";
                $row_array[2]=~ s/\../\t/g;
            }elsif($row_array[2]=~ /^join\((\d+..\d+),(\d+..\d+)\)$/) {
                $row_array[2]="+\t$1\t$row_array[1]\t+\t$2\t$row_array[1]";
                $row_array[2]=~ s/\../\t/g;
            }elsif($row_array[2]=~ /^join\((\d+..\d+),(\d+..\d+),(\d+..\d+)\)$/) {
                $row_array[2]="+\t$1\t$row_array[1]\t+\t$2\t$row_array[1]\t+\t$3\t$row_array[1]";
                $row_array[2]=~ s/\../\t/g;
            }elsif($row_array[2]=~ /^join\(complement\((\d+..\d+)\),complement\((\d+..\d+)\)\)$/){
                $row_array[2]="-\t$1\t$row_array[1]\t-\t$2\t$row_array[1]";
                $row_array[2]=~ s/\../\t/g;
            }elsif($row_array[2]=~ /^join\(complement\((\d+..\d+)\),complement\((\d+..\d+)\),complement\((\d+..\d+)\)\)$/){
                $row_array[2]="-\t$1\t$row_array[1]\t-\t$2\t$row_array[1]\t-\t$3\t$row_array[1]";
                $row_array[2]=~ s/\../\t/g;
            }elsif($row_array[2]=~ /^complement\(join\((\d+..\d+),(\d+..\d+)\)\)$/){
                $row_array[2]="-\t$1\t$row_array[1]\t-\t$2\t$row_array[1]";
                $row_array[2]=~ s/\../\t/g;
            }elsif($row_array[2]=~ /^complement\(join\((\d+..\d+),(\d+..\d+),(\d+..\d+)\)\)$/){
                $row_array[2]="-\t$1\t$row_array[1]\t-\t$2\t$row_array[1]\t-\t$3\t$row_array[1]";
                $row_array[2]=~ s/\../\t/g;
            }elsif($row_array[2]=~ /^order\((\d+..\d+),(\d+..\d+)\)$/){
                $row_array[2]="+\t$1\t$row_array[1]\t+\t$2\t$row_array[1]";
                $row_array[2]=~ s/\../\t/g;
            }elsif($row_array[2]=~ /^order\((\d+..\d+),(\d+..\d+),(\d+..\d+)\)$/){
                $row_array[2]="+\t$1\t$row_array[1]\t+\t$2\t$row_array[1]\t+\t$3\t$row_array[1]";
                $row_array[2]=~ s/\../\t/g;
            }elsif($row_array[2]=~ /^order\(complement\((\d+..\d+)\),complement\((\d+..\d+)\)\)$/){
                $row_array[2]="-\t$1\t$row_array[1]\t-\t$2\t$row_array[1]";
                $row_array[2]=~ s/\../\t/g;
            }elsif($row_array[2]=~ /^order\(complement\((\d+..\d+)\),complement\((\d+..\d+)\),complement\((\d+..\d+)\)\)$/){
                $row_array[2]="-\t$1\t$row_array[1]\t-\t$2\t$row_array[1]\t-\t$3\t$row_array[1]";
                $row_array[2]=~ s/\../\t/g;
            }elsif($row_array[2]=~ /^order\(complement\((\d+..\d+)\),(\d+..\d+)\)$/){
                $row_array[2]="-\t$1\t$row_array[1]\t+\t$2\t$row_array[1]";
                $row_array[2]=~ s/\../\t/g;
            }elsif($row_array[2]=~ /^order\(complement\((\d+..\d+)\),(\d+..\d+),(\d+..\d+)\)$/){
                $row_array[2]="-\t$1\t$row_array[1]\t+\t$2\t$row_array[1]\t+\t$3\t$row_array[1]";
                $row_array[2]=~ s/\../\t/g;
            }elsif($row_array[2]=~ /^join\(complement\((\d+..\d+)\),(\d+..\d+),(\d+..\d+)\)$/){
                $row_array[2]="-\t$1\t$row_array[1]\t+\t$2\t$row_array[1]\t+\t$3\t$row_array[1]";
                $row_array[2]=~ s/\../\t/g;
            }elsif($row_array[2]=~ /^join\((\d+..\d+),(\d+..\d+),complement\((\d+..\d+)\)\)$/) {
                $row_array[2]="+\t$1\t$row_array[1]\t+\t$2\t$row_array[1]\t-\t$3\t$row_array[1]";
                $row_array[2]=~ s/\../\t/g;
            }
            $element=$row_array[2];
            $mark=1;
        }elsif(/^\s+\/gene="(.*)"/ and $mark == 1){
            $element=$species_name.":".$1.":".$element;
            push @genearray,$element;
            $element=();
            $mark=0;
        }
    }
    close $in_genbank;
    foreach (@genearray){
        my @array=split /:/,$_;
        push @output1,"$array[0]\t$array[1]\t$array[2]\n";
    }
    @row_array=();
    @genearray=();


    #put_fasta_sequence_in_array
    my $flag=0;
    my @sequence;
    my (@fas1,@fas2);
    open(my $in_genebank,"<","$filename_base\_temp2");
    while (<$in_genebank>){
        if ($_=~ /ORIGIN/){
            $flag=1;
        }
        if ($_=~ /\/\//){
            $flag=2;
        }
        if ($flag==1){
            next if ($_=~ /ORIGIN/);
            push @sequence,$_;
        }
    }
    close $in_genebank;
    foreach (@sequence){
        chomp;
        $_=~ s/\s*//g;
        $_=~ s/\d+//g;
        push @fas1,$_;
    }
    my $fas1=join "",@fas1;
    my (@fasta1,@fasta2);
    push @fasta1,$species_name,$fas1;
    @fasta2=@fasta1;

    unlink "$filename_base\_temp1";
    unlink "$filename_base\_temp2";


    #edit_bed_file
    my (%SP1,%GENE1,%STRAND1,%START1,%END1,%TYPE1,%STRAND2,%START2,%END2,%TYPE2,%STRAND3,%START3,%END3,%TYPE3,@output2);
    my $cnt1=0;
    foreach (@output1) {
        chomp;
        $cnt1++;
        ($SP1{$cnt1},$GENE1{$cnt1},$STRAND1{$cnt1},$START1{$cnt1},$END1{$cnt1},$TYPE1{$cnt1},$STRAND2{$cnt1},$START2{$cnt1},$END2{$cnt1},$TYPE2{$cnt1},$STRAND3{$cnt1},$START3{$cnt1},$END3{$cnt1},$TYPE3{$cnt1})=(split /\s+/,$_)[0,1,2,3,4,5,6,7,8,9,10,11,12,13];
    }

    foreach (1..$cnt1) {
        if (defined $STRAND2{$_} eq "") {
            push @output2,($SP1{$_}."\t".$GENE1{$_}."\t".$STRAND1{$_}."\t".$START1{$_}."\t".$END1{$_}."\t".$TYPE1{$_});
        }elsif ((defined $STRAND2{$_} ne "") and (defined $STRAND3{$_} eq "")) {
            if (($STRAND1{$_} eq "-") and ($START1{$_} < $START2{$_})){
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-2"."\t".$STRAND1{$_}."\t".$START1{$_}."\t".$END1{$_}."\t".$TYPE1{$_});
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-1"."\t".$STRAND2{$_}."\t".$START2{$_}."\t".$END2{$_}."\t".$TYPE2{$_});
            }elsif(($STRAND1{$_} eq "-") and ($START1{$_} > $START2{$_})){
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-1"."\t".$STRAND1{$_}."\t".$START1{$_}."\t".$END1{$_}."\t".$TYPE1{$_});
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-2"."\t".$STRAND2{$_}."\t".$START2{$_}."\t".$END2{$_}."\t".$TYPE2{$_});
            }elsif(($STRAND1{$_} eq "+") and ($START1{$_} < $START2{$_})){
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-1"."\t".$STRAND1{$_}."\t".$START1{$_}."\t".$END1{$_}."\t".$TYPE1{$_});
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-2"."\t".$STRAND2{$_}."\t".$START2{$_}."\t".$END2{$_}."\t".$TYPE2{$_});
            }elsif(($STRAND1{$_} eq "+") and ($START1{$_} > $START2{$_})){
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-2"."\t".$STRAND1{$_}."\t".$START1{$_}."\t".$END1{$_}."\t".$TYPE1{$_});
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-1"."\t".$STRAND2{$_}."\t".$START2{$_}."\t".$END2{$_}."\t".$TYPE2{$_});
            }
        }elsif ((defined $STRAND2{$_} ne "") and (defined $STRAND3{$_} ne "")) {
            if (($STRAND1{$_} eq "-") and ($START1{$_} < $START2{$_}) and ($START2{$_} < $START3{$_})){
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-3"."\t".$STRAND1{$_}."\t".$START1{$_}."\t".$END1{$_}."\t".$TYPE1{$_});
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-2"."\t".$STRAND2{$_}."\t".$START2{$_}."\t".$END2{$_}."\t".$TYPE2{$_});
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-1"."\t".$STRAND3{$_}."\t".$START3{$_}."\t".$END3{$_}."\t".$TYPE3{$_});
            }elsif(($STRAND1{$_} eq "-") and ($START1{$_} > $START2{$_}) and ($START2{$_} > $START3{$_})){
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-1"."\t".$STRAND1{$_}."\t".$START1{$_}."\t".$END1{$_}."\t".$TYPE1{$_});
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-2"."\t".$STRAND2{$_}."\t".$START2{$_}."\t".$END2{$_}."\t".$TYPE2{$_});
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-3"."\t".$STRAND3{$_}."\t".$START3{$_}."\t".$END3{$_}."\t".$TYPE3{$_});
            }elsif(($STRAND1{$_} eq "-") and ($START1{$_} > $START3{$_}) and ($START3{$_} > $START2{$_})){
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-1"."\t".$STRAND1{$_}."\t".$START1{$_}."\t".$END1{$_}."\t".$TYPE1{$_});
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-2"."\t".$STRAND2{$_}."\t".$START2{$_}."\t".$END2{$_}."\t".$TYPE2{$_});
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-3"."\t".$STRAND3{$_}."\t".$START3{$_}."\t".$END3{$_}."\t".$TYPE3{$_});
            }elsif(($STRAND1{$_} eq "+") and ($START1{$_} < $START2{$_}) and ($START2{$_} < $START3{$_})){
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-1"."\t".$STRAND1{$_}."\t".$START1{$_}."\t".$END1{$_}."\t".$TYPE1{$_});
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-2"."\t".$STRAND2{$_}."\t".$START2{$_}."\t".$END2{$_}."\t".$TYPE2{$_});
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-3"."\t".$STRAND3{$_}."\t".$START3{$_}."\t".$END3{$_}."\t".$TYPE3{$_});
            }elsif(($STRAND1{$_} eq "+") and ($START1{$_} > $START2{$_}) and ($START2{$_} > $START3{$_})){
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-3"."\t".$STRAND1{$_}."\t".$START1{$_}."\t".$END1{$_}."\t".$TYPE1{$_});
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-2"."\t".$STRAND2{$_}."\t".$START2{$_}."\t".$END2{$_}."\t".$TYPE2{$_});
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-1"."\t".$STRAND3{$_}."\t".$START3{$_}."\t".$END3{$_}."\t".$TYPE3{$_});
            }elsif(($STRAND1{$_} eq "+") and ($START1{$_} > $START3{$_}) and ($START3{$_} > $START2{$_})){
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-1"."\t".$STRAND1{$_}."\t".$START1{$_}."\t".$END1{$_}."\t".$TYPE1{$_});
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-2"."\t".$STRAND2{$_}."\t".$START2{$_}."\t".$END2{$_}."\t".$TYPE2{$_});
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-3"."\t".$STRAND3{$_}."\t".$START3{$_}."\t".$END3{$_}."\t".$TYPE3{$_});
            }
        }
    }
    @output1=();


    #sort_bed_file
    my $col=3;
    my (%sort,@output3);
    foreach (@output2){
        my @row=split /\t/,$_;
        $sort{$_}=$row[$col];
    }
    foreach (sort {$sort{$a} <=> $sort{$b}} keys %sort){
        push @output3,"$_\n";
    }
    @output2=();


    #output_bed_file
    open (my $out_bed,">","$filename_base\_bed_coding_noncoding.txt");
    foreach (@output3){
        print $out_bed $_;
    }
    close $out_bed;


    #extract_coding_regions
    my (%SP2,%GENE2,%STRAND4,%START4,%END4,%TYPE4,$seq1);
    my $cnt2=0;
    open(my $out_coding,">","$filename_base\_coding.fasta");
    while (@fasta1){
        my $header=shift @fasta1;
        $seq1=shift @fasta1;
    }
    foreach (@output3){
        chomp;
        $cnt2++;
        ($SP2{$cnt2},$GENE2{$cnt2},$STRAND4{$cnt2},$START4{$cnt2},$END4{$cnt2},$TYPE4{$cnt2})=(split /\s+/,$_)[0,1,2,3,4,5];
        my $str=substr($seq1,($START4{$cnt2}-1),($END4{$cnt2}-$START4{$cnt2}+1));
        if ($STRAND4{$cnt2} eq "-") {
            my $rev_com=reverse $str;
            $rev_com=~ tr/ACGTacgt/TGCAtgca/;
            print $out_coding ">".$GENE2{$cnt2}."_".$SP2{$cnt2}."\n".$rev_com."\n";
        }elsif($STRAND4{$cnt2} eq "+"){
            print $out_coding ">".$GENE2{$cnt2}."_".$SP2{$cnt2}."\n".$str."\n";
        }
    }
    close $out_coding;


    #generate_noncoding_ranges
    my (%SP3,%GENE3,%STRAND5,%START5,%END5,%TYPE5,$last,@output4);
    my $cnt3=0;
    foreach (@output3){
        chomp;
        $cnt3++;
        ($SP3{$cnt3},$GENE3{$cnt3},$STRAND5{$cnt3},$START5{$cnt3},$END5{$cnt3},$TYPE5{$cnt3})=(split /\s+/,$_)[0,1,2,3,4,5];
    }
    foreach (keys %SP3){
        if ($_==1 and $START5{$_}!=1){
            unshift @output4,$SP3{$_}."\t"."start".'-'.$GENE3{$_}."\t"."?"."/".$STRAND5{$_}."\t"."1"."\t".($START5{$_}-1)."\t"."?"."/".$TYPE5{$_}."\n";
        }
    }
    foreach (1..($cnt3-1)) {
        $last=$_+1;
        next if ($END5{$_}+1) >= ($START5{$last}-1);
        push @output4,$SP3{$_}."\t".$GENE3{$_}.'-'.$GENE3{$last}."\t".$STRAND5{$_}."/".$STRAND5{$last}."\t".($END5{$_}+1)."\t".($START5{$last}-1)."\t".$TYPE5{$_}."/".$TYPE5{$last}."\n";
    }
    foreach (keys %SP3){
        if ($_==$cnt3){
            push @output4,$SP3{$_}."\t".$GENE3{$_}.'-'."end"."\t".$STRAND5{$_}."/"."?"."\t".($END5{$_}+1)."\t".$length."\t".$TYPE5{$_}."/"."?"."\n";
        }
    }
    @output3=();


    #extract_noncoding_regions
    my (%SP4,%GENE4,%STRAND6,%START6,%END6,%TYPE6,$seq2);
    my $cnt4=0;
    open(my $out_noncoding,">","$filename_base\_noncoding.fasta");
    while (@fasta2){
        my $header=shift @fasta2;
        $seq2=shift @fasta2;
    }
    foreach (@output4){
        chomp;
        $cnt4++;
        ($SP4{$cnt4},$GENE4{$cnt4},$STRAND6{$cnt4},$START6{$cnt4},$END6{$cnt4},$TYPE6{$cnt4})=(split /\s+/,$_)[0,1,2,3,4,5];
        my $str=substr($seq2,($START6{$cnt4}-1),($END6{$cnt4}-$START6{$cnt4}+1));
        print $out_noncoding ">".$GENE4{$cnt4}."_".$SP4{$cnt4}."\n".$str."\n";
    }
    @output4=();
    close $out_noncoding;
    #unlink "$filename_base\_noncoding.fasta";
}

2、文件夹extract_gene_coding_CDS_RNA，包括分别提取脚本

（1）提取基因

基因：包括内含子。
1_extract_bed_gene.pl

#!/usr/bin/perl -w
use strict;
use File::Find;
use Data::Dumper;
$|=1;

print "Please type your input directory:";
my $dirname=;
chomp $dirname;

my $pattern=".gb";
my @filenames;
find(\&target,$dirname);
sub target{
    if (/$pattern/){
        push @filenames,"$File::Find::name";
    }
    return;
}

while (@filenames) {
    my $filename_gb=shift @filenames;
    my $filename_base=$filename_gb;
    $filename_base=~ s/(.*).gb/$1/g;
    my $latin_name=substr ($filename_base,rindex($filename_base,"\/")+1);

    open(my $in_gb,"<",$filename_gb);
    open(my $out_gb,">","$filename_base\_temp1");
    while (<$in_gb>){
        $_=~ s/\r\n/\n/g;
        if ($_=~ /\),\n/){
            $_=~ s/\),\n/\),/g;
        }elsif($_=~ /,\n/){
            $_=~ s/,\n/,/g;
        }
        print $out_gb $_;
    }
    close $in_gb;
    close $out_gb;

    open(my $in_gbk,"<","$filename_base\_temp1");
    open(my $out_gbk,">","$filename_base\_temp2");
    while (<$in_gbk>){
        $_=~ s/,\s+/,/g;
        print $out_gbk $_;
    }
    close $in_gbk;
    close $out_gbk;


    #generate_bed_file
    my (@row_array,$species_name,$length,$element,@genearray,@output1);
    my $mark=0;
    open (my $in_genbank,"<","$filename_base\_temp2");
    while (<$in_genbank>){
        chomp;
        @row_array=split /\s+/,$_;
        if (/^LOCUS/i){
            $species_name=$latin_name;
            $length=$row_array[2];
        }elsif(/ {5}gene {12}/){
            if ($row_array[2]=~ /^\d+..\d+$/){
                $row_array[2]="\+\t$row_array[2]\t$row_array[1]";
                $row_array[2]=~ s/\../\t/g;
            }elsif($row_array[2]=~/^complement\((\d+..\d+)\)$/){
                $row_array[2]="-\t$1\t$row_array[1]";
                $row_array[2]=~ s/\../\t/g;
            }elsif($row_array[2]=~ /^join\((\d+..\d+),(\d+..\d+)\)$/) {
                $row_array[2]="+\t$1\t$row_array[1]\t+\t$2\t$row_array[1]";
                $row_array[2]=~ s/\../\t/g;
            }elsif($row_array[2]=~ /^join\((\d+..\d+),(\d+..\d+),(\d+..\d+)\)$/) {
                $row_array[2]="+\t$1\t$row_array[1]\t+\t$2\t$row_array[1]\t+\t$3\t$row_array[1]";
                $row_array[2]=~ s/\../\t/g;
            }elsif($row_array[2]=~ /^join\(complement\((\d+..\d+)\),complement\((\d+..\d+)\)\)$/){
                $row_array[2]="-\t$1\t$row_array[1]\t-\t$2\t$row_array[1]";
                $row_array[2]=~ s/\../\t/g;
            }elsif($row_array[2]=~ /^join\(complement\((\d+..\d+)\),(\d+..\d+)\)$/){
                $row_array[2]="-\t$1\t$row_array[1]\t+\t$2\t$row_array[1]";
                $row_array[2]=~ s/\../\t/g;
            }elsif($row_array[2]=~ /^join\(complement\((\d+..\d+)\),complement\((\d+..\d+)\),complement\((\d+..\d+)\)\)$/){
                $row_array[2]="-\t$1\t$row_array[1]\t-\t$2\t$row_array[1]\t-\t$3\t$row_array[1]";
                $row_array[2]=~ s/\../\t/g;
            }elsif($row_array[2]=~ /^complement\(join\((\d+..\d+),(\d+..\d+)\)\)$/){
                $row_array[2]="-\t$1\t$row_array[1]\t-\t$2\t$row_array[1]";
                $row_array[2]=~ s/\../\t/g;
            }elsif($row_array[2]=~ /^complement\(join\((\d+..\d+),(\d+..\d+),(\d+..\d+)\)\)$/){
                $row_array[2]="-\t$1\t$row_array[1]\t-\t$2\t$row_array[1]\t-\t$3\t$row_array[1]";
                $row_array[2]=~ s/\../\t/g;
            }elsif($row_array[2]=~ /^order\((\d+..\d+),(\d+..\d+)\)$/){
                $row_array[2]="+\t$1\t$row_array[1]\t+\t$2\t$row_array[1]";
                $row_array[2]=~ s/\../\t/g;
            }elsif($row_array[2]=~ /^order\((\d+..\d+),(\d+..\d+),(\d+..\d+)\)$/){
                $row_array[2]="+\t$1\t$row_array[1]\t+\t$2\t$row_array[1]\t+\t$3\t$row_array[1]";
                $row_array[2]=~ s/\../\t/g;
            }elsif($row_array[2]=~ /^order\(complement\((\d+..\d+)\),complement\((\d+..\d+)\)\)$/){
                $row_array[2]="-\t$1\t$row_array[1]\t-\t$2\t$row_array[1]";
                $row_array[2]=~ s/\../\t/g;
            }elsif($row_array[2]=~ /^order\(complement\((\d+..\d+)\),complement\((\d+..\d+)\),complement\((\d+..\d+)\)\)$/){
                $row_array[2]="-\t$1\t$row_array[1]\t-\t$2\t$row_array[1]\t-\t$3\t$row_array[1]";
                $row_array[2]=~ s/\../\t/g;
            }elsif($row_array[2]=~ /^order\(complement\((\d+..\d+)\),(\d+..\d+)\)$/){
                $row_array[2]="-\t$1\t$row_array[1]\t+\t$2\t$row_array[1]";
                $row_array[2]=~ s/\../\t/g;
            }elsif($row_array[2]=~ /^order\(complement\((\d+..\d+)\),(\d+..\d+),(\d+..\d+)\)$/){
                $row_array[2]="-\t$1\t$row_array[1]\t+\t$2\t$row_array[1]\t+\t$3\t$row_array[1]";
                $row_array[2]=~ s/\../\t/g;
            }elsif($row_array[2]=~ /^join\(complement\((\d+..\d+)\),(\d+..\d+),(\d+..\d+)\)$/){
                $row_array[2]="-\t$1\t$row_array[1]\t+\t$2\t$row_array[1]\t+\t$3\t$row_array[1]";
                $row_array[2]=~ s/\../\t/g;
            }elsif($row_array[2]=~ /^join\((\d+..\d+),(\d+..\d+),complement\((\d+..\d+)\)\)$/) {
                $row_array[2]="+\t$1\t$row_array[1]\t+\t$2\t$row_array[1]\t-\t$3\t$row_array[1]";
                $row_array[2]=~ s/\../\t/g;
            }
            $element=$row_array[2];
            $mark=1;
        }elsif(/^\s+\/gene="(.*)"/ and $mark == 1){
            $element=$species_name.":".$1.":".$element;
            push @genearray,$element;
            $element=();
            $mark=0;
        }
    }
    close $in_genbank;
    foreach (@genearray){
        my @array=split /:/,$_;
        push @output1,"$array[0]\t$array[1]\t$array[2]\n";
    }
    @row_array=();
    @genearray=();


    #put_fasta_sequence_in_array
    my $flag=0;
    my @sequence;
    my (@fas1,@fas2);
    open(my $in_genebank,"<","$filename_base\_temp2");
    while (<$in_genebank>){
        if ($_=~ /ORIGIN/){
            $flag=1;
        }
        if ($_=~ /\/\//){
            $flag=2;
        }
        if ($flag==1){
            next if ($_=~ /ORIGIN/);
            push @sequence,$_;
        }
    }
    close $in_genebank;
    foreach (@sequence){
        chomp;
        $_=~ s/\s*//g;
        $_=~ s/\d+//g;
        push @fas1,$_;
    }
    my $fas1=join "",@fas1;
    my (@fasta1,@fasta2);
    push @fasta1,$species_name,$fas1;
    @fasta2=@fasta1;

    unlink "$filename_base\_temp1";
    unlink "$filename_base\_temp2";


    #edit_bed_file
    my (%SP1,%GENE1,%STRAND1,%START1,%END1,%TYPE1,%STRAND2,%START2,%END2,%TYPE2,%STRAND3,%START3,%END3,%TYPE3,@output2);
    my $cnt1=0;
    foreach (@output1) {
        chomp;
        $cnt1++;
        ($SP1{$cnt1},$GENE1{$cnt1},$STRAND1{$cnt1},$START1{$cnt1},$END1{$cnt1},$TYPE1{$cnt1},$STRAND2{$cnt1},$START2{$cnt1},$END2{$cnt1},$TYPE2{$cnt1})=(split /\s+/,$_)[0,1,2,3,4,5,6,7,8,9];
    }
    foreach (1..$cnt1) {
        if (defined $STRAND2{$_} eq "") {
            push @output2,($SP1{$_}."\t".$GENE1{$_}."\t".$STRAND1{$_}."\t".$START1{$_}."\t".$END1{$_}."\t".$TYPE1{$_});
        }elsif (defined $STRAND2{$_} ne "") {
            if (($STRAND1{$_} eq "-") and ($STRAND2{$_} eq "-") and ($START1{$_} < $START2{$_})){
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-2"."\t".$STRAND1{$_}."\t".$START1{$_}."\t".$END1{$_}."\t".$TYPE1{$_});
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-1"."\t".$STRAND2{$_}."\t".$START2{$_}."\t".$END2{$_}."\t".$TYPE2{$_});
            }elsif(($STRAND1{$_} eq "-") and ($STRAND2{$_} eq "-") and ($START1{$_} > $START2{$_})){
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-1"."\t".$STRAND1{$_}."\t".$START1{$_}."\t".$END1{$_}."\t".$TYPE1{$_});
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-2"."\t".$STRAND2{$_}."\t".$START2{$_}."\t".$END2{$_}."\t".$TYPE2{$_});
            }elsif(($STRAND1{$_} eq "-") and ($STRAND2{$_} eq "+") and ($START1{$_} > $START2{$_})){
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-1"."\t".$STRAND1{$_}."\t".$START1{$_}."\t".$END1{$_}."\t".$TYPE1{$_});
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-2"."\t".$STRAND2{$_}."\t".$START2{$_}."\t".$END2{$_}."\t".$TYPE2{$_});
            }elsif(($STRAND1{$_} eq "+") and ($STRAND2{$_} eq "+") and ($START1{$_} < $START2{$_})){
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-1"."\t".$STRAND1{$_}."\t".$START1{$_}."\t".$END1{$_}."\t".$TYPE1{$_});
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-2"."\t".$STRAND2{$_}."\t".$START2{$_}."\t".$END2{$_}."\t".$TYPE2{$_});
            }elsif(($STRAND1{$_} eq "+") and ($STRAND2{$_} eq "+") and ($START1{$_} > $START2{$_}) and ($GENE1{$_} ne "rps12")){
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-2"."\t".$STRAND1{$_}."\t".$START1{$_}."\t".$END1{$_}."\t".$TYPE1{$_});
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-1"."\t".$STRAND2{$_}."\t".$START2{$_}."\t".$END2{$_}."\t".$TYPE2{$_});
            }elsif(($STRAND1{$_} eq "+") and ($STRAND2{$_} eq "+") and ($START1{$_} > $START2{$_}) and ($GENE1{$_} eq "rps12")){
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-1"."\t".$STRAND1{$_}."\t".$START1{$_}."\t".$END1{$_}."\t".$TYPE1{$_});
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-2"."\t".$STRAND2{$_}."\t".$START2{$_}."\t".$END2{$_}."\t".$TYPE2{$_});
            }
        }
    }
    @output1=();


    #sort_bed_file
    my $col=3;
    my (%sort,@output3);
    foreach (@output2){
        my @row=split /\t/,$_;
        $sort{$_}=$row[$col];
    }
    foreach (sort {$sort{$a} <=> $sort{$b}} keys %sort){
        push @output3,"$_\n";
    }
    @output2=();


    #output_bed_file
    open (my $out_bed,">","$filename_base\_gene.bed");
    foreach (@output3){
        print $out_bed $_;
    }
    close $out_bed;


    #extract_gene
    my (%SP2,%GENE2,%STRAND4,%START4,%END4,%TYPE4,$seq1);
    my $cnt2=0;
    open(my $out_coding,">","$filename_base\_gene.fasta");
    while (@fasta1){
        my $header=shift @fasta1;
        $seq1=shift @fasta1;
    }
    foreach (@output3){
        chomp;
        $cnt2++;
        ($SP2{$cnt2},$GENE2{$cnt2},$STRAND4{$cnt2},$START4{$cnt2},$END4{$cnt2},$TYPE4{$cnt2})=(split /\s+/,$_)[0,1,2,3,4,5];
        my $str=substr($seq1,($START4{$cnt2}-1),($END4{$cnt2}-$START4{$cnt2}+1));
        if ($STRAND4{$cnt2} eq "-") {
            my $rev_com=reverse $str;
            $rev_com=~ tr/ACGTacgt/TGCAtgca/;
            print $out_coding ">".$GENE2{$cnt2}."_".$SP2{$cnt2}."\n".$rev_com."\n";
        }elsif($STRAND4{$cnt2} eq "+"){
            print $out_coding ">".$GENE2{$cnt2}."_".$SP2{$cnt2}."\n".$str."\n";
        }
    }
    close $out_coding;


    #generate_IGS_ranges
    my (%SP3,%GENE3,%STRAND5,%START5,%END5,%TYPE5,$last0,$last1,$last2,@output4);
    my $cnt3=0;
    foreach (@output3){
        chomp;
        $cnt3++;
        ($SP3{$cnt3},$GENE3{$cnt3},$STRAND5{$cnt3},$START5{$cnt3},$END5{$cnt3},$TYPE5{$cnt3})=(split /\s+/,$_)[0,1,2,3,4,5];
    }
    foreach (keys %SP3){
        if ($_==1 and $START5{$_}!=1){
            unshift @output4,$SP3{$_}."\t"."start".'-'.$GENE3{$_}."\t"."?"."/".$STRAND5{$_}."\t"."1"."\t".($START5{$_}-1)."\t"."?"."/".$TYPE5{$_}."\n";
        }
    }
    foreach (1..($cnt3-1)) {
        $last0=$_-1;
        $last1=$_+1;
        $last2=$_+2;
        next if ((($END5{$_}+1) >= ($START5{$last1}-1)) and (($END5{$_}+1) < ($END5{$last1}-1)));
        next if (($_ > 1) and (($END5{$_}+1) < ($END5{$last0}-1)) and (($END5{$_}+1) < ($START5{$last2}-1)));
        if ((($END5{$_}+1) >= ($START5{$last1}-1)) and (($END5{$_}+1) >= ($END5{$last1}-1))){
            push @output4,$SP3{$_}."\t".$GENE3{$_}.'-'.$GENE3{$last2}."\t".$STRAND5{$_}."/".$STRAND5{$last2}."\t".($END5{$_}+1)."\t".($START5{$last2}-1)."\t".$TYPE5{$_}."/".$TYPE5{$last2}."\n";
        }else{
            push @output4,$SP3{$_}."\t".$GENE3{$_}.'-'.$GENE3{$last1}."\t".$STRAND5{$_}."/".$STRAND5{$last1}."\t".($END5{$_}+1)."\t".($START5{$last1}-1)."\t".$TYPE5{$_}."/".$TYPE5{$last1}."\n";
        }
    }
    foreach (keys %SP3){
        if ($_==$cnt3){
            push @output4,$SP3{$_}."\t".$GENE3{$_}.'-'."end"."\t".$STRAND5{$_}."/"."?"."\t".($END5{$_}+1)."\t".$length."\t".$TYPE5{$_}."/"."?"."\n";
        }
    }
    @output3=();


    #extract_IGS
    my (%SP4,%GENE4,%STRAND6,%START6,%END6,%TYPE6,$seq2);
    my $cnt4=0;
    open(my $out_noncoding,">","$filename_base\_IGS.fasta");
    while (@fasta2){
        my $header=shift @fasta2;
        $seq2=shift @fasta2;
    }
    foreach (@output4){
        chomp;
        $cnt4++;
        ($SP4{$cnt4},$GENE4{$cnt4},$STRAND6{$cnt4},$START6{$cnt4},$END6{$cnt4},$TYPE6{$cnt4})=(split /\s+/,$_)[0,1,2,3,4,5];
        my $str=substr($seq2,($START6{$cnt4}-1),($END6{$cnt4}-$START6{$cnt4}+1));
        print $out_noncoding ">".$GENE4{$cnt4}."_".$SP4{$cnt4}."\n".$str."\n";
    }
    @output4=();
    close $out_noncoding;
    unlink "$filename_base\_IGS.fasta";
}

（2）提取基因编码区

基因编码区：外显子单独提取，例如trnK-UUU-2、trnK-UUU-1。
2_extract_bed_coding.pl

#!/usr/bin/perl -w
use strict;
use File::Find;
use Data::Dumper;
$|=1;

print "Please type your input directory:";
my $dirname=;
chomp $dirname;

my $pattern=".gb";
my @filenames;
find(\&target,$dirname);
sub target{
    if (/$pattern/){
        push @filenames,"$File::Find::name";
    }
    return;
}

while (@filenames) {
    my $filename_gb=shift @filenames;
    my $filename_base=$filename_gb;
    $filename_base=~ s/(.*).gb/$1/g;
    my $latin_name=substr ($filename_base,rindex($filename_base,"\/")+1);

    open(my $in_gb,"<",$filename_gb);
    open(my $out_gb,">","$filename_base\_temp1");
    while (<$in_gb>){
        $_=~ s/\r\n/\n/g;
        if ($_=~ /\),\n/){
            $_=~ s/\),\n/\),/g;
        }elsif($_=~ /,\n/){
            $_=~ s/,\n/,/g;
        }
        print $out_gb $_;
    }
    close $in_gb;
    close $out_gb;

    open(my $in_gbk,"<","$filename_base\_temp1");
    open(my $out_gbk,">","$filename_base\_temp2");
    while (<$in_gbk>){
        $_=~ s/,\s+/,/g;
        print $out_gbk $_;
    }
    close $in_gbk;
    close $out_gbk;


    #generate_bed_file
    my (@row_array,$species_name,$length,$element,@genearray,@output1);
    my $mark=0;
    open (my $in_genbank,"<","$filename_base\_temp2");
    while (<$in_genbank>){
        chomp;
        @row_array=split /\s+/,$_;
        if (/^LOCUS/i){
            $species_name=$latin_name;
            $length=$row_array[2];
        }elsif(/ {5}CDS {13}/ or / {5}tRNA {12}/ or / {5}rRNA {12}/){
            if ($row_array[2]=~ /^\d+..\d+$/){
                $row_array[2]="\+\t$row_array[2]\t$row_array[1]";
                $row_array[2]=~ s/\../\t/g;
            }elsif($row_array[2]=~/^complement\((\d+..\d+)\)$/){
                $row_array[2]="-\t$1\t$row_array[1]";
                $row_array[2]=~ s/\../\t/g;
            }elsif($row_array[2]=~ /^join\((\d+..\d+),(\d+..\d+)\)$/) {
                $row_array[2]="+\t$1\t$row_array[1]\t+\t$2\t$row_array[1]";
                $row_array[2]=~ s/\../\t/g;
            }elsif($row_array[2]=~ /^join\((\d+..\d+),(\d+..\d+),(\d+..\d+)\)$/) {
                $row_array[2]="+\t$1\t$row_array[1]\t+\t$2\t$row_array[1]\t+\t$3\t$row_array[1]";
                $row_array[2]=~ s/\../\t/g;
            }elsif($row_array[2]=~ /^join\(complement\((\d+..\d+)\),complement\((\d+..\d+)\)\)$/){
                $row_array[2]="-\t$1\t$row_array[1]\t-\t$2\t$row_array[1]";
                $row_array[2]=~ s/\../\t/g;
            }elsif($row_array[2]=~ /^join\(complement\((\d+..\d+)\),complement\((\d+..\d+)\),complement\((\d+..\d+)\)\)$/){
                $row_array[2]="-\t$1\t$row_array[1]\t-\t$2\t$row_array[1]\t-\t$3\t$row_array[1]";
                $row_array[2]=~ s/\../\t/g;
            }elsif($row_array[2]=~ /^complement\(join\((\d+..\d+),(\d+..\d+)\)\)$/){
                $row_array[2]="-\t$1\t$row_array[1]\t-\t$2\t$row_array[1]";
                $row_array[2]=~ s/\../\t/g;
            }elsif($row_array[2]=~ /^complement\(join\((\d+..\d+),(\d+..\d+),(\d+..\d+)\)\)$/){
                $row_array[2]="-\t$1\t$row_array[1]\t-\t$2\t$row_array[1]\t-\t$3\t$row_array[1]";
                $row_array[2]=~ s/\../\t/g;
            }elsif($row_array[2]=~ /^order\((\d+..\d+),(\d+..\d+)\)$/){
                $row_array[2]="+\t$1\t$row_array[1]\t+\t$2\t$row_array[1]";
                $row_array[2]=~ s/\../\t/g;
            }elsif($row_array[2]=~ /^order\((\d+..\d+),(\d+..\d+),(\d+..\d+)\)$/){
                $row_array[2]="+\t$1\t$row_array[1]\t+\t$2\t$row_array[1]\t+\t$3\t$row_array[1]";
                $row_array[2]=~ s/\../\t/g;
            }elsif($row_array[2]=~ /^order\(complement\((\d+..\d+)\),complement\((\d+..\d+)\)\)$/){
                $row_array[2]="-\t$1\t$row_array[1]\t-\t$2\t$row_array[1]";
                $row_array[2]=~ s/\../\t/g;
            }elsif($row_array[2]=~ /^order\(complement\((\d+..\d+)\),complement\((\d+..\d+)\),complement\((\d+..\d+)\)\)$/){
                $row_array[2]="-\t$1\t$row_array[1]\t-\t$2\t$row_array[1]\t-\t$3\t$row_array[1]";
                $row_array[2]=~ s/\../\t/g;
            }elsif($row_array[2]=~ /^order\(complement\((\d+..\d+)\),(\d+..\d+)\)$/){
                $row_array[2]="-\t$1\t$row_array[1]\t+\t$2\t$row_array[1]";
                $row_array[2]=~ s/\../\t/g;
            }elsif($row_array[2]=~ /^order\(complement\((\d+..\d+)\),(\d+..\d+),(\d+..\d+)\)$/){
                $row_array[2]="-\t$1\t$row_array[1]\t+\t$2\t$row_array[1]\t+\t$3\t$row_array[1]";
                $row_array[2]=~ s/\../\t/g;
            }elsif($row_array[2]=~ /^join\(complement\((\d+..\d+)\),(\d+..\d+),(\d+..\d+)\)$/){
                $row_array[2]="-\t$1\t$row_array[1]\t+\t$2\t$row_array[1]\t+\t$3\t$row_array[1]";
                $row_array[2]=~ s/\../\t/g;
            }elsif($row_array[2]=~ /^join\((\d+..\d+),(\d+..\d+),complement\((\d+..\d+)\)\)$/) {
                $row_array[2]="+\t$1\t$row_array[1]\t+\t$2\t$row_array[1]\t-\t$3\t$row_array[1]";
                $row_array[2]=~ s/\../\t/g;
            }
            $element=$row_array[2];
            $mark=1;
        }elsif(/^\s+\/gene="(.*)"/ and $mark == 1){
            $element=$species_name.":".$1.":".$element;
            push @genearray,$element;
            $element=();
            $mark=0;
        }
    }
    close $in_genbank;
    foreach (@genearray){
        my @array=split /:/,$_;
        push @output1,"$array[0]\t$array[1]\t$array[2]\n";
    }
    @row_array=();
    @genearray=();


    #put_fasta_sequence_in_array
    my $flag=0;
    my @sequence;
    my (@fas1,@fas2);
    open(my $in_genebank,"<","$filename_base\_temp2");
    while (<$in_genebank>){
        if ($_=~ /ORIGIN/){
            $flag=1;
        }
        if ($_=~ /\/\//){
            $flag=2;
        }
        if ($flag==1){
            next if ($_=~ /ORIGIN/);
            push @sequence,$_;
        }
    }
    close $in_genebank;
    foreach (@sequence){
        chomp;
        $_=~ s/\s*//g;
        $_=~ s/\d+//g;
        push @fas1,$_;
    }
    my $fas1=join "",@fas1;
    my (@fasta1,@fasta2);
    push @fasta1,$species_name,$fas1;
    @fasta2=@fasta1;

    unlink "$filename_base\_temp1";
    unlink "$filename_base\_temp2";


    #edit_bed_file
    my (%SP1,%GENE1,%STRAND1,%START1,%END1,%TYPE1,%STRAND2,%START2,%END2,%TYPE2,%STRAND3,%START3,%END3,%TYPE3,@output2);
    my $cnt1=0;
    foreach (@output1) {
        chomp;
        $cnt1++;
        ($SP1{$cnt1},$GENE1{$cnt1},$STRAND1{$cnt1},$START1{$cnt1},$END1{$cnt1},$TYPE1{$cnt1},$STRAND2{$cnt1},$START2{$cnt1},$END2{$cnt1},$TYPE2{$cnt1},$STRAND3{$cnt1},$START3{$cnt1},$END3{$cnt1},$TYPE3{$cnt1})=(split /\s+/,$_)[0,1,2,3,4,5,6,7,8,9,10,11,12,13];
    }

    foreach (1..$cnt1) {
        if (defined $STRAND2{$_} eq "") {
            push @output2,($SP1{$_}."\t".$GENE1{$_}."\t".$STRAND1{$_}."\t".$START1{$_}."\t".$END1{$_}."\t".$TYPE1{$_});
        }elsif ((defined $STRAND2{$_} ne "") and (defined $STRAND3{$_} eq "")) {
            if (($STRAND1{$_} eq "-") and ($START1{$_} < $START2{$_})){
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-2"."\t".$STRAND1{$_}."\t".$START1{$_}."\t".$END1{$_}."\t".$TYPE1{$_});
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-1"."\t".$STRAND2{$_}."\t".$START2{$_}."\t".$END2{$_}."\t".$TYPE2{$_});
            }elsif(($STRAND1{$_} eq "-") and ($START1{$_} > $START2{$_})){
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-1"."\t".$STRAND1{$_}."\t".$START1{$_}."\t".$END1{$_}."\t".$TYPE1{$_});
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-2"."\t".$STRAND2{$_}."\t".$START2{$_}."\t".$END2{$_}."\t".$TYPE2{$_});
            }elsif(($STRAND1{$_} eq "+") and ($START1{$_} < $START2{$_})){
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-1"."\t".$STRAND1{$_}."\t".$START1{$_}."\t".$END1{$_}."\t".$TYPE1{$_});
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-2"."\t".$STRAND2{$_}."\t".$START2{$_}."\t".$END2{$_}."\t".$TYPE2{$_});
            }elsif(($STRAND1{$_} eq "+") and ($START1{$_} > $START2{$_})){
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-2"."\t".$STRAND1{$_}."\t".$START1{$_}."\t".$END1{$_}."\t".$TYPE1{$_});
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-1"."\t".$STRAND2{$_}."\t".$START2{$_}."\t".$END2{$_}."\t".$TYPE2{$_});
            }
        }elsif ((defined $STRAND2{$_} ne "") and (defined $STRAND3{$_} ne "")) {
            if (($STRAND1{$_} eq "-") and ($START1{$_} < $START2{$_}) and ($START2{$_} < $START3{$_})){
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-3"."\t".$STRAND1{$_}."\t".$START1{$_}."\t".$END1{$_}."\t".$TYPE1{$_});
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-2"."\t".$STRAND2{$_}."\t".$START2{$_}."\t".$END2{$_}."\t".$TYPE2{$_});
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-1"."\t".$STRAND3{$_}."\t".$START3{$_}."\t".$END3{$_}."\t".$TYPE3{$_});
            }elsif(($STRAND1{$_} eq "-") and ($START1{$_} > $START2{$_}) and ($START2{$_} > $START3{$_})){
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-1"."\t".$STRAND1{$_}."\t".$START1{$_}."\t".$END1{$_}."\t".$TYPE1{$_});
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-2"."\t".$STRAND2{$_}."\t".$START2{$_}."\t".$END2{$_}."\t".$TYPE2{$_});
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-3"."\t".$STRAND3{$_}."\t".$START3{$_}."\t".$END3{$_}."\t".$TYPE3{$_});
            }elsif(($STRAND1{$_} eq "-") and ($START1{$_} > $START3{$_}) and ($START3{$_} > $START2{$_})){
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-1"."\t".$STRAND1{$_}."\t".$START1{$_}."\t".$END1{$_}."\t".$TYPE1{$_});
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-2"."\t".$STRAND2{$_}."\t".$START2{$_}."\t".$END2{$_}."\t".$TYPE2{$_});
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-3"."\t".$STRAND3{$_}."\t".$START3{$_}."\t".$END3{$_}."\t".$TYPE3{$_});
            }elsif(($STRAND1{$_} eq "+") and ($START1{$_} < $START2{$_}) and ($START2{$_} < $START3{$_})){
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-1"."\t".$STRAND1{$_}."\t".$START1{$_}."\t".$END1{$_}."\t".$TYPE1{$_});
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-2"."\t".$STRAND2{$_}."\t".$START2{$_}."\t".$END2{$_}."\t".$TYPE2{$_});
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-3"."\t".$STRAND3{$_}."\t".$START3{$_}."\t".$END3{$_}."\t".$TYPE3{$_});
            }elsif(($STRAND1{$_} eq "+") and ($START1{$_} > $START2{$_}) and ($START2{$_} > $START3{$_})){
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-3"."\t".$STRAND1{$_}."\t".$START1{$_}."\t".$END1{$_}."\t".$TYPE1{$_});
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-2"."\t".$STRAND2{$_}."\t".$START2{$_}."\t".$END2{$_}."\t".$TYPE2{$_});
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-1"."\t".$STRAND3{$_}."\t".$START3{$_}."\t".$END3{$_}."\t".$TYPE3{$_});
            }elsif(($STRAND1{$_} eq "+") and ($START1{$_} > $START3{$_}) and ($START3{$_} > $START2{$_})){
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-1"."\t".$STRAND1{$_}."\t".$START1{$_}."\t".$END1{$_}."\t".$TYPE1{$_});
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-2"."\t".$STRAND2{$_}."\t".$START2{$_}."\t".$END2{$_}."\t".$TYPE2{$_});
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-3"."\t".$STRAND3{$_}."\t".$START3{$_}."\t".$END3{$_}."\t".$TYPE3{$_});
            }
        }
    }
    @output1=();


    #sort_bed_file
    my $col=3;
    my (%sort,@output3);
    foreach (@output2){
        my @row=split /\t/,$_;
        $sort{$_}=$row[$col];
    }
    foreach (sort {$sort{$a} <=> $sort{$b}} keys %sort){
        push @output3,"$_\n";
    }
    @output2=();


    #output_bed_file
    open (my $out_bed,">","$filename_base\_coding.bed");
    foreach (@output3){
        print $out_bed $_;
    }
    close $out_bed;


    #extract_coding_regions
    my (%SP2,%GENE2,%STRAND4,%START4,%END4,%TYPE4,$seq1);
    my $cnt2=0;
    open(my $out_coding,">","$filename_base\_coding.fasta");
    while (@fasta1){
        my $header=shift @fasta1;
        $seq1=shift @fasta1;
    }
    foreach (@output3){
        chomp;
        $cnt2++;
        ($SP2{$cnt2},$GENE2{$cnt2},$STRAND4{$cnt2},$START4{$cnt2},$END4{$cnt2},$TYPE4{$cnt2})=(split /\s+/,$_)[0,1,2,3,4,5];
        my $str=substr($seq1,($START4{$cnt2}-1),($END4{$cnt2}-$START4{$cnt2}+1));
        if ($STRAND4{$cnt2} eq "-") {
            my $rev_com=reverse $str;
            $rev_com=~ tr/ACGTacgt/TGCAtgca/;
            print $out_coding ">".$GENE2{$cnt2}."_".$SP2{$cnt2}."\n".$rev_com."\n";
        }elsif($STRAND4{$cnt2} eq "+"){
            print $out_coding ">".$GENE2{$cnt2}."_".$SP2{$cnt2}."\n".$str."\n";
        }
    }
    close $out_coding;


    #generate_noncoding_ranges
    my (%SP3,%GENE3,%STRAND5,%START5,%END5,%TYPE5,$last,@output4);
    my $cnt3=0;
    foreach (@output3){
        chomp;
        $cnt3++;
        ($SP3{$cnt3},$GENE3{$cnt3},$STRAND5{$cnt3},$START5{$cnt3},$END5{$cnt3},$TYPE5{$cnt3})=(split /\s+/,$_)[0,1,2,3,4,5];
    }
    foreach (keys %SP3){
        if ($_==1 and $START5{$_}!=1){
            unshift @output4,$SP3{$_}."\t"."start".'-'.$GENE3{$_}."\t"."?"."/".$STRAND5{$_}."\t"."1"."\t".($START5{$_}-1)."\t"."?"."/".$TYPE5{$_}."\n";
        }
    }
    foreach (1..($cnt3-1)) {
        $last=$_+1;
        next if ($END5{$_}+1) >= ($START5{$last}-1);
        push @output4,$SP3{$_}."\t".$GENE3{$_}.'-'.$GENE3{$last}."\t".$STRAND5{$_}."/".$STRAND5{$last}."\t".($END5{$_}+1)."\t".($START5{$last}-1)."\t".$TYPE5{$_}."/".$TYPE5{$last}."\n";
    }
    foreach (keys %SP3){
        if ($_==$cnt3){
            push @output4,$SP3{$_}."\t".$GENE3{$_}.'-'."end"."\t".$STRAND5{$_}."/"."?"."\t".($END5{$_}+1)."\t".$length."\t".$TYPE5{$_}."/"."?"."\n";
        }
    }
    @output3=();


    #extract_noncoding_regions
    my (%SP4,%GENE4,%STRAND6,%START6,%END6,%TYPE6,$seq2);
    my $cnt4=0;
    open(my $out_noncoding,">","$filename_base\_noncoding.fasta");
    while (@fasta2){
        my $header=shift @fasta2;
        $seq2=shift @fasta2;
    }
    foreach (@output4){
        chomp;
        $cnt4++;
        ($SP4{$cnt4},$GENE4{$cnt4},$STRAND6{$cnt4},$START6{$cnt4},$END6{$cnt4},$TYPE6{$cnt4})=(split /\s+/,$_)[0,1,2,3,4,5];
        my $str=substr($seq2,($START6{$cnt4}-1),($END6{$cnt4}-$START6{$cnt4}+1));
        print $out_noncoding ">".$GENE4{$cnt4}."_".$SP4{$cnt4}."\n".$str."\n";
    }
    @output4=();
    close $out_noncoding;
    unlink "$filename_base\_noncoding.fasta";
}

（3）提取CDS（蛋白）编码基因

CDS（蛋白）编码基因：不包括内含子，外显子合并。
3_extract_bed_CDS.pl

#!/usr/bin/perl -w
use strict;
use File::Find;
use Data::Dumper;
$|=1;

print "Please type your input directory:";
my $dirname=;
chomp $dirname;

my $pattern=".gb";
my @filenames;
find(\&target,$dirname);
sub target{
    if (/$pattern/){
        push @filenames,"$File::Find::name";
    }
    return;
}

while (@filenames) {
    my $filename_gb=shift @filenames;
    my $filename_base=$filename_gb;
    $filename_base=~ s/(.*).gb/$1/g;
    my $latin_name=substr ($filename_base,rindex($filename_base,"\/")+1);

    open(my $in_gb,"<",$filename_gb);
    open(my $out_gb,">","$filename_base\_temp1");
    while (<$in_gb>){
        $_=~ s/\r\n/\n/g;
        if ($_=~ /\),\n/){
            $_=~ s/\),\n/\),/g;
        }elsif($_=~ /,\n/){
            $_=~ s/,\n/,/g;
        }
        print $out_gb $_;
    }
    close $in_gb;
    close $out_gb;

    open(my $in_gbk,"<","$filename_base\_temp1");
    open(my $out_gbk,">","$filename_base\_temp2");
    while (<$in_gbk>){
        $_=~ s/,\s+/,/g;
        print $out_gbk $_;
    }
    close $in_gbk;
    close $out_gbk;


    #generate_bed_file
    my (@row_array,$species_name,$length,$element,@genearray,@output1);
    my $mark=0;
    open (my $in_genbank,"<","$filename_base\_temp2");
    while (<$in_genbank>){
        chomp;
        @row_array=split /\s+/,$_;
        if (/^LOCUS/i){
            $species_name=$latin_name;
            $length=$row_array[2];
        }elsif(/ {5}CDS {13}/){
            if ($row_array[2]=~ /^\d+..\d+$/){
                $row_array[2]="\+\t$row_array[2]\t$row_array[1]";
                $row_array[2]=~ s/\../\t/g;
            }elsif($row_array[2]=~/^complement\((\d+..\d+)\)$/){
                $row_array[2]="-\t$1\t$row_array[1]";
                $row_array[2]=~ s/\../\t/g;
            }elsif($row_array[2]=~ /^join\((\d+..\d+),(\d+..\d+)\)$/) {
                $row_array[2]="+\t$1\t$row_array[1]\t+\t$2\t$row_array[1]";
                $row_array[2]=~ s/\../\t/g;
            }elsif($row_array[2]=~ /^join\((\d+..\d+),(\d+..\d+),(\d+..\d+)\)$/) {
                $row_array[2]="+\t$1\t$row_array[1]\t+\t$2\t$row_array[1]\t+\t$3\t$row_array[1]";
                $row_array[2]=~ s/\../\t/g;
            }elsif($row_array[2]=~ /^join\(complement\((\d+..\d+)\),complement\((\d+..\d+)\)\)$/){
                $row_array[2]="-\t$1\t$row_array[1]\t-\t$2\t$row_array[1]";
                $row_array[2]=~ s/\../\t/g;
            }elsif($row_array[2]=~ /^join\(complement\((\d+..\d+)\),complement\((\d+..\d+)\),complement\((\d+..\d+)\)\)$/){
                $row_array[2]="-\t$1\t$row_array[1]\t-\t$2\t$row_array[1]\t-\t$3\t$row_array[1]";
                $row_array[2]=~ s/\../\t/g;
            }elsif($row_array[2]=~ /^complement\(join\((\d+..\d+),(\d+..\d+)\)\)$/){
                $row_array[2]="-\t$1\t$row_array[1]\t-\t$2\t$row_array[1]";
                $row_array[2]=~ s/\../\t/g;
            }elsif($row_array[2]=~ /^complement\(join\((\d+..\d+),(\d+..\d+),(\d+..\d+)\)\)$/){
                $row_array[2]="-\t$1\t$row_array[1]\t-\t$2\t$row_array[1]\t-\t$3\t$row_array[1]";
                $row_array[2]=~ s/\../\t/g;
            }elsif($row_array[2]=~ /^order\((\d+..\d+),(\d+..\d+)\)$/){
                $row_array[2]="+\t$1\t$row_array[1]\t+\t$2\t$row_array[1]";
                $row_array[2]=~ s/\../\t/g;
            }elsif($row_array[2]=~ /^order\((\d+..\d+),(\d+..\d+),(\d+..\d+)\)$/){
                $row_array[2]="+\t$1\t$row_array[1]\t+\t$2\t$row_array[1]\t+\t$3\t$row_array[1]";
                $row_array[2]=~ s/\../\t/g;
            }elsif($row_array[2]=~ /^order\(complement\((\d+..\d+)\),complement\((\d+..\d+)\)\)$/){
                $row_array[2]="-\t$1\t$row_array[1]\t-\t$2\t$row_array[1]";
                $row_array[2]=~ s/\../\t/g;
            }elsif($row_array[2]=~ /^order\(complement\((\d+..\d+)\),complement\((\d+..\d+)\),complement\((\d+..\d+)\)\)$/){
                $row_array[2]="-\t$1\t$row_array[1]\t-\t$2\t$row_array[1]\t-\t$3\t$row_array[1]";
                $row_array[2]=~ s/\../\t/g;
            }elsif($row_array[2]=~ /^order\(complement\((\d+..\d+)\),(\d+..\d+)\)$/){
                $row_array[2]="-\t$1\t$row_array[1]\t+\t$2\t$row_array[1]";
                $row_array[2]=~ s/\../\t/g;
            }elsif($row_array[2]=~ /^order\(complement\((\d+..\d+)\),(\d+..\d+),(\d+..\d+)\)$/){
                $row_array[2]="-\t$1\t$row_array[1]\t+\t$2\t$row_array[1]\t+\t$3\t$row_array[1]";
                $row_array[2]=~ s/\../\t/g;
            }elsif($row_array[2]=~ /^join\(complement\((\d+..\d+)\),(\d+..\d+),(\d+..\d+)\)$/){
                $row_array[2]="-\t$1\t$row_array[1]\t+\t$2\t$row_array[1]\t+\t$3\t$row_array[1]";
                $row_array[2]=~ s/\../\t/g;
            }elsif($row_array[2]=~ /^join\((\d+..\d+),(\d+..\d+),complement\((\d+..\d+)\)\)$/) {
                $row_array[2]="+\t$1\t$row_array[1]\t+\t$2\t$row_array[1]\t-\t$3\t$row_array[1]";
                $row_array[2]=~ s/\../\t/g;
            }
            $element=$row_array[2];
            $mark=1;
        }elsif(/^\s+\/gene="(.*)"/ and $mark == 1){
            $element=$species_name.":".$1.":".$element;
            push @genearray,$element;
            $element=();
            $mark=0;
        }
    }
    close $in_genbank;
    foreach (@genearray){
        my @array=split /:/,$_;
        push @output1,"$array[0]\t$array[1]\t$array[2]\n";
    }
    @row_array=();
    @genearray=();


    #put_fasta_sequence_in_array
    my $flag=0;
    my @sequence;
    my (@fas1,@fas2);
    open(my $in_genebank,"<","$filename_base\_temp2");
    while (<$in_genebank>){
        if ($_=~ /ORIGIN/){
            $flag=1;
        }
        if ($_=~ /\/\//){
            $flag=2;
        }
        if ($flag==1){
            next if ($_=~ /ORIGIN/);
            push @sequence,$_;
        }
    }
    close $in_genebank;
    foreach (@sequence){
        chomp;
        $_=~ s/\s*//g;
        $_=~ s/\d+//g;
        push @fas1,$_;
    }
    my $fas1=join "",@fas1;
    my (@fasta1,@fasta2);
    push @fasta1,$species_name,$fas1;
    @fasta2=@fasta1;

    unlink "$filename_base\_temp1";
    unlink "$filename_base\_temp2";


    #edit_bed_file
    my (%SP1,%GENE1,%STRAND1,%START1,%END1,%TYPE1,%STRAND2,%START2,%END2,%TYPE2,%STRAND3,%START3,%END3,%TYPE3,@output2);
    my $cnt1=0;
    foreach (@output1) {
        chomp;
        $cnt1++;
        ($SP1{$cnt1},$GENE1{$cnt1},$STRAND1{$cnt1},$START1{$cnt1},$END1{$cnt1},$TYPE1{$cnt1},$STRAND2{$cnt1},$START2{$cnt1},$END2{$cnt1},$TYPE2{$cnt1},$STRAND3{$cnt1},$START3{$cnt1},$END3{$cnt1},$TYPE3{$cnt1})=(split /\s+/,$_)[0,1,2,3,4,5,6,7,8,9,10,11,12,13];
    }

    foreach (1..$cnt1) {
        if (defined $STRAND2{$_} eq "") {
            push @output2,($SP1{$_}."\t".$GENE1{$_}."\t".$STRAND1{$_}."\t".$START1{$_}."\t".$END1{$_}."\t".$TYPE1{$_});
        }elsif ((defined $STRAND2{$_} ne "") and (defined $STRAND3{$_} eq "")) {
            if (($STRAND1{$_} eq "-") and ($START1{$_} < $START2{$_})){
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-2"."\t".$STRAND1{$_}."\t".$START1{$_}."\t".$END1{$_}."\t".$TYPE1{$_});
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-1"."\t".$STRAND2{$_}."\t".$START2{$_}."\t".$END2{$_}."\t".$TYPE2{$_});
            }elsif(($STRAND1{$_} eq "-") and ($START1{$_} > $START2{$_})){
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-1"."\t".$STRAND1{$_}."\t".$START1{$_}."\t".$END1{$_}."\t".$TYPE1{$_});
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-2"."\t".$STRAND2{$_}."\t".$START2{$_}."\t".$END2{$_}."\t".$TYPE2{$_});
            }elsif(($STRAND1{$_} eq "+") and ($START1{$_} < $START2{$_})){
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-1"."\t".$STRAND1{$_}."\t".$START1{$_}."\t".$END1{$_}."\t".$TYPE1{$_});
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-2"."\t".$STRAND2{$_}."\t".$START2{$_}."\t".$END2{$_}."\t".$TYPE2{$_});
            }elsif(($STRAND1{$_} eq "+") and ($START1{$_} > $START2{$_})){
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-2"."\t".$STRAND1{$_}."\t".$START1{$_}."\t".$END1{$_}."\t".$TYPE1{$_});
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-1"."\t".$STRAND2{$_}."\t".$START2{$_}."\t".$END2{$_}."\t".$TYPE2{$_});
            }
        }elsif ((defined $STRAND2{$_} ne "") and (defined $STRAND3{$_} ne "")) {
            if (($STRAND1{$_} eq "-") and ($START1{$_} < $START2{$_}) and ($START2{$_} < $START3{$_})){
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-3"."\t".$STRAND1{$_}."\t".$START1{$_}."\t".$END1{$_}."\t".$TYPE1{$_});
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-2"."\t".$STRAND2{$_}."\t".$START2{$_}."\t".$END2{$_}."\t".$TYPE2{$_});
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-1"."\t".$STRAND3{$_}."\t".$START3{$_}."\t".$END3{$_}."\t".$TYPE3{$_});
            }elsif(($STRAND1{$_} eq "-") and ($START1{$_} > $START2{$_}) and ($START2{$_} > $START3{$_})){
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-1"."\t".$STRAND1{$_}."\t".$START1{$_}."\t".$END1{$_}."\t".$TYPE1{$_});
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-2"."\t".$STRAND2{$_}."\t".$START2{$_}."\t".$END2{$_}."\t".$TYPE2{$_});
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-3"."\t".$STRAND3{$_}."\t".$START3{$_}."\t".$END3{$_}."\t".$TYPE3{$_});
            }elsif(($STRAND1{$_} eq "-") and ($START1{$_} > $START3{$_}) and ($START3{$_} > $START2{$_})){
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-1"."\t".$STRAND1{$_}."\t".$START1{$_}."\t".$END1{$_}."\t".$TYPE1{$_});
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-2"."\t".$STRAND2{$_}."\t".$START2{$_}."\t".$END2{$_}."\t".$TYPE2{$_});
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-3"."\t".$STRAND3{$_}."\t".$START3{$_}."\t".$END3{$_}."\t".$TYPE3{$_});
            }elsif(($STRAND1{$_} eq "+") and ($START1{$_} < $START2{$_}) and ($START2{$_} < $START3{$_})){
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-1"."\t".$STRAND1{$_}."\t".$START1{$_}."\t".$END1{$_}."\t".$TYPE1{$_});
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-2"."\t".$STRAND2{$_}."\t".$START2{$_}."\t".$END2{$_}."\t".$TYPE2{$_});
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-3"."\t".$STRAND3{$_}."\t".$START3{$_}."\t".$END3{$_}."\t".$TYPE3{$_});
            }elsif(($STRAND1{$_} eq "+") and ($START1{$_} > $START2{$_}) and ($START2{$_} > $START3{$_})){
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-3"."\t".$STRAND1{$_}."\t".$START1{$_}."\t".$END1{$_}."\t".$TYPE1{$_});
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-2"."\t".$STRAND2{$_}."\t".$START2{$_}."\t".$END2{$_}."\t".$TYPE2{$_});
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-1"."\t".$STRAND3{$_}."\t".$START3{$_}."\t".$END3{$_}."\t".$TYPE3{$_});
            }elsif(($STRAND1{$_} eq "+") and ($START1{$_} > $START3{$_}) and ($START3{$_} > $START2{$_})){
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-1"."\t".$STRAND1{$_}."\t".$START1{$_}."\t".$END1{$_}."\t".$TYPE1{$_});
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-2"."\t".$STRAND2{$_}."\t".$START2{$_}."\t".$END2{$_}."\t".$TYPE2{$_});
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-3"."\t".$STRAND3{$_}."\t".$START3{$_}."\t".$END3{$_}."\t".$TYPE3{$_});
            }
        }
    }


    #sort_bed_file
    my $col=3;
    my (%sort,@output3);
    foreach (@output2){
        my @row=split /\t/,$_;
        $sort{$_}=$row[$col];
    }
    foreach (sort {$sort{$a} <=> $sort{$b}} keys %sort){
        push @output3,"$_\n";
    }
    @output2=();


    #output_bed_file
    open (my $out_bed,">","$filename_base\_CDS.bed");
    foreach (@output3){
        print $out_bed $_;
    }
    close $out_bed;


    #extract_CDS
    my (%SP2,%GENE2,%STRAND4,%START4,%END4,%TYPE4,%STRAND5,%START5,%END5,%TYPE5,%STRAND6,%START6,%END6,%TYPE6,$seq1);
    my $cnt2=0;
    open(my $out_coding,">","$filename_base\_CDS.fasta");
    while (@fasta1){
        my $header=shift @fasta1;
        $seq1=shift @fasta1;
    }
    foreach (@output1){
        chomp;
        $cnt2++;
        ($SP2{$cnt2},$GENE2{$cnt2},$STRAND4{$cnt2},$START4{$cnt2},$END4{$cnt2},$TYPE4{$cnt2},$STRAND5{$cnt2},$START5{$cnt2},$END5{$cnt2},$TYPE5{$cnt2},$STRAND6{$cnt2},$START6{$cnt2},$END6{$cnt2},$TYPE6{$cnt2})=(split /\s+/,$_)[0,1,2,3,4,5,6,7,8,9,10,11,12,13];
        if (defined $STRAND5{$cnt2} eq "") {
            my $str1=substr($seq1,($START4{$cnt2}-1),($END4{$cnt2}-$START4{$cnt2}+1));
            if ($STRAND4{$cnt2} eq "-") {
                my $rev_com1=reverse $str1;
                $rev_com1=~ tr/ACGTacgt/TGCAtgca/;
                print $out_coding ">".$GENE2{$cnt2}."_".$SP2{$cnt2}."\n".$rev_com1."\n";
            }elsif($STRAND4{$cnt2} eq "+"){
                print $out_coding ">".$GENE2{$cnt2}."_".$SP2{$cnt2}."\n".$str1."\n";
            }
        }elsif((defined $STRAND5{$cnt2} ne "") and (defined $STRAND6{$cnt2} eq "")) {
            if (($STRAND4{$cnt2} eq "-") and ($START4{$cnt2} < $START5{$cnt2})) {
                my $str2=substr($seq1,($START4{$cnt2}-1),($END4{$cnt2}-$START4{$cnt2}+1)).substr($seq1,($START5{$cnt2}-1),($END5{$cnt2}-$START5{$cnt2}+1));
                my $rev_com2=reverse $str2;
                $rev_com2=~ tr/ACGTacgt/TGCAtgca/;
                print $out_coding ">".$GENE2{$cnt2}."_".$SP2{$cnt2}."\n".$rev_com2."\n";
            }elsif(($STRAND4{$cnt2} eq "-") and ($START4{$cnt2} > $START5{$cnt2})) {
                my $str3=substr($seq1,($START5{$cnt2}-1),($END5{$cnt2}-$START5{$cnt2}+1)).substr($seq1,($START4{$cnt2}-1),($END4{$cnt2}-$START4{$cnt2}+1));
                my $rev_com3=reverse $str3;
                $rev_com3=~ tr/ACGTacgt/TGCAtgca/;
                print $out_coding ">".$GENE2{$cnt2}."_".$SP2{$cnt2}."\n".$rev_com3."\n";
            }elsif(($STRAND4{$cnt2} eq "+") and ($START4{$cnt2} < $START5{$cnt2})){
                my $str4=substr($seq1,($START4{$cnt2}-1),($END4{$cnt2}-$START4{$cnt2}+1)).substr($seq1,($START5{$cnt2}-1),($END5{$cnt2}-$START5{$cnt2}+1));
                print $out_coding ">".$GENE2{$cnt2}."_".$SP2{$cnt2}."\n".$str4."\n";
            }elsif(($STRAND4{$cnt2} eq "+") and ($START4{$cnt2} > $START5{$cnt2})){
                my $str5=substr($seq1,($START5{$cnt2}-1),($END5{$cnt2}-$START5{$cnt2}+1)).substr($seq1,($START4{$cnt2}-1),($END4{$cnt2}-$START4{$cnt2}+1));
                print $out_coding ">".$GENE2{$cnt2}."_".$SP2{$cnt2}."\n".$str5."\n";
            }
        }elsif ((defined $STRAND5{$cnt2} ne "") and (defined $STRAND6{$cnt2} ne "")) {
            if (($STRAND4{$cnt2} eq "-") and ($START4{$cnt2} < $START5{$cnt2}) and ($START5{$cnt2} < $START6{$cnt2})) {
                my $str6=substr($seq1,($START4{$cnt2}-1),($END4{$cnt2}-$START4{$cnt2}+1)).substr($seq1,($START5{$cnt2}-1),($END5{$cnt2}-$START5{$cnt2}+1)).substr($seq1,($START6{$cnt2}-1),($END6{$cnt2}-$START6{$cnt2}+1));
                my $rev_com4=reverse $str6;
                $rev_com4=~ tr/ACGTacgt/TGCAtgca/;
                print $out_coding ">".$GENE2{$cnt2}."_".$SP2{$cnt2}."\n".$rev_com4."\n";
            }elsif(($STRAND4{$cnt2} eq "-") and ($START4{$cnt2} > $START5{$cnt2}) and ($START5{$cnt2} > $START6{$cnt2})) {
                my $str7=substr($seq1,($START6{$cnt2}-1),($END6{$cnt2}-$START6{$cnt2}+1)).substr($seq1,($START5{$cnt2}-1),($END5{$cnt2}-$START5{$cnt2}+1)).substr($seq1,($START4{$cnt2}-1),($END4{$cnt2}-$START4{$cnt2}+1));
                my $rev_com5=reverse $str7;
                $rev_com5=~ tr/ACGTacgt/TGCAtgca/;
                print $out_coding ">".$GENE2{$cnt2}."_".$SP2{$cnt2}."\n".$rev_com5."\n";
            }elsif(($STRAND4{$cnt2} eq "-") and ($START4{$cnt2} > $START6{$cnt2}) and ($START6{$cnt2} > $START5{$cnt2})) {
                my $str8=substr($seq1,($START4{$cnt2}-1),($END4{$cnt2}-$START4{$cnt2}+1));
                my $str9=substr($seq1,($START5{$cnt2}-1),($END5{$cnt2}-$START5{$cnt2}+1)).substr($seq1,($START6{$cnt2}-1),($END6{$cnt2}-$START6{$cnt2}+1));
                my $rev_com6=reverse $str8;
                $rev_com6=~ tr/ACGTacgt/TGCAtgca/;
                print $out_coding ">".$GENE2{$cnt2}."_".$SP2{$cnt2}."\n".$rev_com6.$str9."\n";
            }elsif(($STRAND4{$cnt2} eq "+") and ($START4{$cnt2} < $START5{$cnt2}) and ($START5{$cnt2} < $START6{$cnt2})){
                my $str10=substr($seq1,($START4{$cnt2}-1),($END4{$cnt2}-$START4{$cnt2}+1)).substr($seq1,($START5{$cnt2}-1),($END5{$cnt2}-$START5{$cnt2}+1)).substr($seq1,($START6{$cnt2}-1),($END6{$cnt2}-$START6{$cnt2}+1));
                print $out_coding ">".$GENE2{$cnt2}."_".$SP2{$cnt2}."\n".$str10."\n";
            }elsif(($STRAND4{$cnt2} eq "+") and ($START4{$cnt2} > $START5{$cnt2}) and ($START5{$cnt2} > $START6{$cnt2})){
                my $str11=substr($seq1,($START6{$cnt2}-1),($END6{$cnt2}-$START6{$cnt2}+1)).substr($seq1,($START5{$cnt2}-1),($END5{$cnt2}-$START5{$cnt2}+1)).substr($seq1,($START4{$cnt2}-1),($END4{$cnt2}-$START4{$cnt2}+1));
                print $out_coding ">".$GENE2{$cnt2}."_".$SP2{$cnt2}."\n".$str11."\n";
            }elsif(($STRAND4{$cnt2} eq "+") and ($START4{$cnt2} > $START6{$cnt2}) and ($START6{$cnt2} > $START5{$cnt2})) {
                my $str12=substr($seq1,($START4{$cnt2}-1),($END4{$cnt2}-$START4{$cnt2}+1)).substr($seq1,($START5{$cnt2}-1),($END5{$cnt2}-$START5{$cnt2}+1)).substr($seq1,($START6{$cnt2}-1),($END6{$cnt2}-$START6{$cnt2}+1));
                print $out_coding ">".$GENE2{$cnt2}."_".$SP2{$cnt2}."\n".$str12."\n";
            }
        }
    }
    close $out_coding;
    @output1=();


    #generate_intergenic_ranges
    my (%SP3,%GENE3,%STRAND7,%START7,%END7,%TYPE7,$last0,$last1,$last2,@output4);
    my $cnt3=0;
    foreach (@output3){
        chomp;
        $cnt3++;
        ($SP3{$cnt3},$GENE3{$cnt3},$STRAND7{$cnt3},$START7{$cnt3},$END7{$cnt3},$TYPE7{$cnt3})=(split /\s+/,$_)[0,1,2,3,4,5];
    }
    foreach (keys %SP3){
        if ($_==1 and $START7{$_}!=1){
            unshift @output4,$SP3{$_}."\t"."start".'-'.$GENE3{$_}."\t"."?"."/".$STRAND7{$_}."\t"."1"."\t".($START7{$_}-1)."\t"."?"."/".$TYPE7{$_}."\n";
        }
    }
    foreach (1..($cnt3-1)) {
        $last0=$_-1;
        $last1=$_+1;
        $last2=$_+2;
        next if ((($END7{$_}+1) >= ($START7{$last1}-1)) and (($END7{$_}+1) < ($END7{$last1}-1)));
        next if (($_ > 1) and (($END7{$_}+1) < ($END7{$last0}-1)) and (($END7{$_}+1) < ($START7{$last2}-1)));
        if ((($END7{$_}+1) >= ($START7{$last1}-1)) and (($END7{$_}+1) >= ($END7{$last1}-1))){
            push @output4,$SP3{$_}."\t".$GENE3{$_}.'-'.$GENE3{$last2}."\t".$STRAND7{$_}."/".$STRAND7{$last2}."\t".($END7{$_}+1)."\t".($START7{$last2}-1)."\t".$TYPE7{$_}."/".$TYPE7{$last2}."\n";
        }else{
            push @output4,$SP3{$_}."\t".$GENE3{$_}.'-'.$GENE3{$last1}."\t".$STRAND7{$_}."/".$STRAND7{$last1}."\t".($END7{$_}+1)."\t".($START7{$last1}-1)."\t".$TYPE7{$_}."/".$TYPE7{$last1}."\n";
        }
    }
    foreach (keys %SP3){
        if ($_==$cnt3){
            push @output4,$SP3{$_}."\t".$GENE3{$_}.'-'."end"."\t".$STRAND7{$_}."/"."?"."\t".($END7{$_}+1)."\t".$length."\t".$TYPE7{$_}."/"."?"."\n";
        }
    }
    @output3=();


    #extract_intergenic
    my (%SP4,%GENE4,%STRAND8,%START8,%END8,%TYPE8,$seq2);
    my $cnt4=0;
    open(my $out_noncoding,">","$filename_base\_intergenic.fasta");
    while (@fasta2){
        my $header=shift @fasta2;
        $seq2=shift @fasta2;
    }
    foreach (@output4){
        chomp;
        $cnt4++;
        ($SP4{$cnt4},$GENE4{$cnt4},$STRAND8{$cnt4},$START8{$cnt4},$END8{$cnt4},$TYPE8{$cnt4})=(split /\s+/,$_)[0,1,2,3,4,5];
        my $str=substr($seq2,($START8{$cnt4}-1),($END8{$cnt4}-$START8{$cnt4}+1));
        print $out_noncoding ">".$GENE4{$cnt4}."_".$SP4{$cnt4}."\n".$str."\n";
    }
    @output4=();
    close $out_noncoding;
    unlink "$filename_base\_intergenic.fasta";
}

（4）提取RNA编码基因

RNA编码基因：不包括内含子，外显子合并。
4_extract_bed_RNA.pl

#!/usr/bin/perl -w
use strict;
use File::Find;
use Data::Dumper;
$|=1;

print "Please type your input directory:";
my $dirname=;
chomp $dirname;

my $pattern=".gb";
my @filenames;
find(\&target,$dirname);
sub target{
    if (/$pattern/){
        push @filenames,"$File::Find::name";
    }
    return;
}

while (@filenames) {
    my $filename_gb=shift @filenames;
    my $filename_base=$filename_gb;
    $filename_base=~ s/(.*).gb/$1/g;
    my $latin_name=substr ($filename_base,rindex($filename_base,"\/")+1);

    open(my $in_gb,"<",$filename_gb);
    open(my $out_gb,">","$filename_base\_temp1");
    while (<$in_gb>){
        $_=~ s/\r\n/\n/g;
        if ($_=~ /\),\n/){
            $_=~ s/\),\n/\),/g;
        }elsif($_=~ /,\n/){
            $_=~ s/,\n/,/g;
        }
        print $out_gb $_;
    }
    close $in_gb;
    close $out_gb;

    open(my $in_gbk,"<","$filename_base\_temp1");
    open(my $out_gbk,">","$filename_base\_temp2");
    while (<$in_gbk>){
        $_=~ s/,\s+/,/g;
        print $out_gbk $_;
    }
    close $in_gbk;
    close $out_gbk;


    #generate_bed_file
    my (@row_array,$species_name,$length,$element,@genearray,@output1);
    my $mark=0;
    open (my $in_genbank,"<","$filename_base\_temp2");
    while (<$in_genbank>){
        chomp;
        @row_array=split /\s+/,$_;
        if (/^LOCUS/i){
            $species_name=$latin_name;
            $length=$row_array[2];
        }elsif(/ {5}tRNA {12}/ or / {5}rRNA {12}/){
            if ($row_array[2]=~ /^\d+..\d+$/){
                $row_array[2]="\+\t$row_array[2]\t$row_array[1]";
                $row_array[2]=~ s/\../\t/g;
            }elsif($row_array[2]=~/^complement\((\d+..\d+)\)$/){
                $row_array[2]="-\t$1\t$row_array[1]";
                $row_array[2]=~ s/\../\t/g;
            }elsif($row_array[2]=~ /^join\((\d+..\d+),(\d+..\d+)\)$/) {
                $row_array[2]="+\t$1\t$row_array[1]\t+\t$2\t$row_array[1]";
                $row_array[2]=~ s/\../\t/g;
            }elsif($row_array[2]=~ /^join\((\d+..\d+),(\d+..\d+),(\d+..\d+)\)$/) {
                $row_array[2]="+\t$1\t$row_array[1]\t+\t$2\t$row_array[1]\t+\t$3\t$row_array[1]";
                $row_array[2]=~ s/\../\t/g;
            }elsif($row_array[2]=~ /^join\(complement\((\d+..\d+)\),complement\((\d+..\d+)\)\)$/){
                $row_array[2]="-\t$1\t$row_array[1]\t-\t$2\t$row_array[1]";
                $row_array[2]=~ s/\../\t/g;
            }elsif($row_array[2]=~ /^join\(complement\((\d+..\d+)\),complement\((\d+..\d+)\),complement\((\d+..\d+)\)\)$/){
                $row_array[2]="-\t$1\t$row_array[1]\t-\t$2\t$row_array[1]\t-\t$3\t$row_array[1]";
                $row_array[2]=~ s/\../\t/g;
            }elsif($row_array[2]=~ /^complement\(join\((\d+..\d+),(\d+..\d+)\)\)$/){
                $row_array[2]="-\t$1\t$row_array[1]\t-\t$2\t$row_array[1]";
                $row_array[2]=~ s/\../\t/g;
            }elsif($row_array[2]=~ /^complement\(join\((\d+..\d+),(\d+..\d+),(\d+..\d+)\)\)$/){
                $row_array[2]="-\t$1\t$row_array[1]\t-\t$2\t$row_array[1]\t-\t$3\t$row_array[1]";
                $row_array[2]=~ s/\../\t/g;
            }elsif($row_array[2]=~ /^order\((\d+..\d+),(\d+..\d+)\)$/){
                $row_array[2]="+\t$1\t$row_array[1]\t+\t$2\t$row_array[1]";
                $row_array[2]=~ s/\../\t/g;
            }elsif($row_array[2]=~ /^order\((\d+..\d+),(\d+..\d+),(\d+..\d+)\)$/){
                $row_array[2]="+\t$1\t$row_array[1]\t+\t$2\t$row_array[1]\t+\t$3\t$row_array[1]";
                $row_array[2]=~ s/\../\t/g;
            }elsif($row_array[2]=~ /^order\(complement\((\d+..\d+)\),complement\((\d+..\d+)\)\)$/){
                $row_array[2]="-\t$1\t$row_array[1]\t-\t$2\t$row_array[1]";
                $row_array[2]=~ s/\../\t/g;
            }elsif($row_array[2]=~ /^order\(complement\((\d+..\d+)\),complement\((\d+..\d+)\),complement\((\d+..\d+)\)\)$/){
                $row_array[2]="-\t$1\t$row_array[1]\t-\t$2\t$row_array[1]\t-\t$3\t$row_array[1]";
                $row_array[2]=~ s/\../\t/g;
            }elsif($row_array[2]=~ /^order\(complement\((\d+..\d+)\),(\d+..\d+)\)$/){
                $row_array[2]="-\t$1\t$row_array[1]\t+\t$2\t$row_array[1]";
                $row_array[2]=~ s/\../\t/g;
            }elsif($row_array[2]=~ /^order\(complement\((\d+..\d+)\),(\d+..\d+),(\d+..\d+)\)$/){
                $row_array[2]="-\t$1\t$row_array[1]\t+\t$2\t$row_array[1]\t+\t$3\t$row_array[1]";
                $row_array[2]=~ s/\../\t/g;
            }elsif($row_array[2]=~ /^join\(complement\((\d+..\d+)\),(\d+..\d+),(\d+..\d+)\)$/){
                $row_array[2]="-\t$1\t$row_array[1]\t+\t$2\t$row_array[1]\t+\t$3\t$row_array[1]";
                $row_array[2]=~ s/\../\t/g;
            }elsif($row_array[2]=~ /^join\((\d+..\d+),(\d+..\d+),complement\((\d+..\d+)\)\)$/) {
                $row_array[2]="+\t$1\t$row_array[1]\t+\t$2\t$row_array[1]\t-\t$3\t$row_array[1]";
                $row_array[2]=~ s/\../\t/g;
            }
            $element=$row_array[2];
            $mark=1;
        }elsif(/^\s+\/gene="(.*)"/ and $mark == 1){
            $element=$species_name.":".$1.":".$element;
            push @genearray,$element;
            $element=();
            $mark=0;
        }
    }
    close $in_genbank;
    foreach (@genearray){
        my @array=split /:/,$_;
        push @output1,"$array[0]\t$array[1]\t$array[2]\n";
    }
    @row_array=();
    @genearray=();


    #put_fasta_sequence_in_array
    my $flag=0;
    my @sequence;
    my (@fas1,@fas2);
    open(my $in_genebank,"<","$filename_base\_temp2");
    while (<$in_genebank>){
        if ($_=~ /ORIGIN/){
            $flag=1;
        }
        if ($_=~ /\/\//){
            $flag=2;
        }
        if ($flag==1){
            next if ($_=~ /ORIGIN/);
            push @sequence,$_;
        }
    }
    close $in_genebank;
    foreach (@sequence){
        chomp;
        $_=~ s/\s*//g;
        $_=~ s/\d+//g;
        push @fas1,$_;
    }
    my $fas1=join "",@fas1;
    my (@fasta1,@fasta2);
    push @fasta1,$species_name,$fas1;
    @fasta2=@fasta1;

    unlink "$filename_base\_temp1";
    unlink "$filename_base\_temp2";


    #edit_bed_file
    my (%SP1,%GENE1,%STRAND1,%START1,%END1,%TYPE1,%STRAND2,%START2,%END2,%TYPE2,%STRAND3,%START3,%END3,%TYPE3,@output2);
    my $cnt1=0;
    foreach (@output1) {
        chomp;
        $cnt1++;
        ($SP1{$cnt1},$GENE1{$cnt1},$STRAND1{$cnt1},$START1{$cnt1},$END1{$cnt1},$TYPE1{$cnt1},$STRAND2{$cnt1},$START2{$cnt1},$END2{$cnt1},$TYPE2{$cnt1},$STRAND3{$cnt1},$START3{$cnt1},$END3{$cnt1},$TYPE3{$cnt1})=(split /\s+/,$_)[0,1,2,3,4,5,6,7,8,9,10,11,12,13];
    }

    foreach (1..$cnt1) {
        if (defined $STRAND2{$_} eq "") {
            push @output2,($SP1{$_}."\t".$GENE1{$_}."\t".$STRAND1{$_}."\t".$START1{$_}."\t".$END1{$_}."\t".$TYPE1{$_});
        }elsif ((defined $STRAND2{$_} ne "") and (defined $STRAND3{$_} eq "")) {
            if (($STRAND1{$_} eq "-") and ($START1{$_} < $START2{$_})){
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-2"."\t".$STRAND1{$_}."\t".$START1{$_}."\t".$END1{$_}."\t".$TYPE1{$_});
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-1"."\t".$STRAND2{$_}."\t".$START2{$_}."\t".$END2{$_}."\t".$TYPE2{$_});
            }elsif(($STRAND1{$_} eq "-") and ($START1{$_} > $START2{$_})){
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-1"."\t".$STRAND1{$_}."\t".$START1{$_}."\t".$END1{$_}."\t".$TYPE1{$_});
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-2"."\t".$STRAND2{$_}."\t".$START2{$_}."\t".$END2{$_}."\t".$TYPE2{$_});
            }elsif(($STRAND1{$_} eq "+") and ($START1{$_} < $START2{$_})){
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-1"."\t".$STRAND1{$_}."\t".$START1{$_}."\t".$END1{$_}."\t".$TYPE1{$_});
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-2"."\t".$STRAND2{$_}."\t".$START2{$_}."\t".$END2{$_}."\t".$TYPE2{$_});
            }elsif(($STRAND1{$_} eq "+") and ($START1{$_} > $START2{$_})){
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-2"."\t".$STRAND1{$_}."\t".$START1{$_}."\t".$END1{$_}."\t".$TYPE1{$_});
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-1"."\t".$STRAND2{$_}."\t".$START2{$_}."\t".$END2{$_}."\t".$TYPE2{$_});
            }
        }elsif ((defined $STRAND2{$_} ne "") and (defined $STRAND3{$_} ne "")) {
            if (($STRAND1{$_} eq "-") and ($START1{$_} < $START2{$_}) and ($START2{$_} < $START3{$_})){
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-3"."\t".$STRAND1{$_}."\t".$START1{$_}."\t".$END1{$_}."\t".$TYPE1{$_});
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-2"."\t".$STRAND2{$_}."\t".$START2{$_}."\t".$END2{$_}."\t".$TYPE2{$_});
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-1"."\t".$STRAND3{$_}."\t".$START3{$_}."\t".$END3{$_}."\t".$TYPE3{$_});
            }elsif(($STRAND1{$_} eq "-") and ($START1{$_} > $START2{$_}) and ($START2{$_} > $START3{$_})){
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-1"."\t".$STRAND1{$_}."\t".$START1{$_}."\t".$END1{$_}."\t".$TYPE1{$_});
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-2"."\t".$STRAND2{$_}."\t".$START2{$_}."\t".$END2{$_}."\t".$TYPE2{$_});
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-3"."\t".$STRAND3{$_}."\t".$START3{$_}."\t".$END3{$_}."\t".$TYPE3{$_});
            }elsif(($STRAND1{$_} eq "-") and ($START1{$_} > $START3{$_}) and ($START3{$_} > $START2{$_})){
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-1"."\t".$STRAND1{$_}."\t".$START1{$_}."\t".$END1{$_}."\t".$TYPE1{$_});
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-2"."\t".$STRAND2{$_}."\t".$START2{$_}."\t".$END2{$_}."\t".$TYPE2{$_});
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-3"."\t".$STRAND3{$_}."\t".$START3{$_}."\t".$END3{$_}."\t".$TYPE3{$_});
            }elsif(($STRAND1{$_} eq "+") and ($START1{$_} < $START2{$_}) and ($START2{$_} < $START3{$_})){
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-1"."\t".$STRAND1{$_}."\t".$START1{$_}."\t".$END1{$_}."\t".$TYPE1{$_});
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-2"."\t".$STRAND2{$_}."\t".$START2{$_}."\t".$END2{$_}."\t".$TYPE2{$_});
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-3"."\t".$STRAND3{$_}."\t".$START3{$_}."\t".$END3{$_}."\t".$TYPE3{$_});
            }elsif(($STRAND1{$_} eq "+") and ($START1{$_} > $START2{$_}) and ($START2{$_} > $START3{$_})){
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-3"."\t".$STRAND1{$_}."\t".$START1{$_}."\t".$END1{$_}."\t".$TYPE1{$_});
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-2"."\t".$STRAND2{$_}."\t".$START2{$_}."\t".$END2{$_}."\t".$TYPE2{$_});
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-1"."\t".$STRAND3{$_}."\t".$START3{$_}."\t".$END3{$_}."\t".$TYPE3{$_});
            }elsif(($STRAND1{$_} eq "+") and ($START1{$_} > $START3{$_}) and ($START3{$_} > $START2{$_})){
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-1"."\t".$STRAND1{$_}."\t".$START1{$_}."\t".$END1{$_}."\t".$TYPE1{$_});
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-2"."\t".$STRAND2{$_}."\t".$START2{$_}."\t".$END2{$_}."\t".$TYPE2{$_});
                push @output2,($SP1{$_}."\t".$GENE1{$_}."-3"."\t".$STRAND3{$_}."\t".$START3{$_}."\t".$END3{$_}."\t".$TYPE3{$_});
            }
        }
    }


    #sort_bed_file
    my $col=3;
    my (%sort,@output3);
    foreach (@output2){
        my @row=split /\t/,$_;
        $sort{$_}=$row[$col];
    }
    foreach (sort {$sort{$a} <=> $sort{$b}} keys %sort){
        push @output3,"$_\n";
    }
    @output2=();


    #output_bed_file
    open (my $out_bed,">","$filename_base\_RNA.bed");
    foreach (@output3){
        print $out_bed $_;
    }
    close $out_bed;


    #extract_gene
    my (%SP2,%GENE2,%STRAND4,%START4,%END4,%TYPE4,%STRAND5,%START5,%END5,%TYPE5,%STRAND6,%START6,%END6,%TYPE6,$seq1);
    my $cnt2=0;
    open(my $out_coding,">","$filename_base\_RNA.fasta");
    while (@fasta1){
        my $header=shift @fasta1;
        $seq1=shift @fasta1;
    }
    foreach (@output1){
        chomp;
        $cnt2++;
        ($SP2{$cnt2},$GENE2{$cnt2},$STRAND4{$cnt2},$START4{$cnt2},$END4{$cnt2},$TYPE4{$cnt2},$STRAND5{$cnt2},$START5{$cnt2},$END5{$cnt2},$TYPE5{$cnt2},$STRAND6{$cnt2},$START6{$cnt2},$END6{$cnt2},$TYPE6{$cnt2})=(split /\s+/,$_)[0,1,2,3,4,5,6,7,8,9,10,11,12,13];
        if (defined $STRAND5{$cnt2} eq "") {
            my $str1=substr($seq1,($START4{$cnt2}-1),($END4{$cnt2}-$START4{$cnt2}+1));
            if ($STRAND4{$cnt2} eq "-") {
                my $rev_com1=reverse $str1;
                $rev_com1=~ tr/ACGTacgt/TGCAtgca/;
                print $out_coding ">".$GENE2{$cnt2}."_".$SP2{$cnt2}."\n".$rev_com1."\n";
            }elsif($STRAND4{$cnt2} eq "+"){
                print $out_coding ">".$GENE2{$cnt2}."_".$SP2{$cnt2}."\n".$str1."\n";
            }
        }elsif((defined $STRAND5{$cnt2} ne "") and (defined $STRAND6{$cnt2} eq "")) {
            if (($STRAND4{$cnt2} eq "-") and ($START4{$cnt2} < $START5{$cnt2})) {
                my $str2=substr($seq1,($START4{$cnt2}-1),($END4{$cnt2}-$START4{$cnt2}+1)).substr($seq1,($START5{$cnt2}-1),($END5{$cnt2}-$START5{$cnt2}+1));
                my $rev_com2=reverse $str2;
                $rev_com2=~ tr/ACGTacgt/TGCAtgca/;
                print $out_coding ">".$GENE2{$cnt2}."_".$SP2{$cnt2}."\n".$rev_com2."\n";
            }elsif(($STRAND4{$cnt2} eq "-") and ($START4{$cnt2} > $START5{$cnt2})) {
                my $str3=substr($seq1,($START5{$cnt2}-1),($END5{$cnt2}-$START5{$cnt2}+1)).substr($seq1,($START4{$cnt2}-1),($END4{$cnt2}-$START4{$cnt2}+1));
                my $rev_com3=reverse $str3;
                $rev_com3=~ tr/ACGTacgt/TGCAtgca/;
                print $out_coding ">".$GENE2{$cnt2}."_".$SP2{$cnt2}."\n".$rev_com3."\n";
            }elsif(($STRAND4{$cnt2} eq "+") and ($START4{$cnt2} < $START5{$cnt2})){
                my $str4=substr($seq1,($START4{$cnt2}-1),($END4{$cnt2}-$START4{$cnt2}+1)).substr($seq1,($START5{$cnt2}-1),($END5{$cnt2}-$START5{$cnt2}+1));
                print $out_coding ">".$GENE2{$cnt2}."_".$SP2{$cnt2}."\n".$str4."\n";
            }elsif(($STRAND4{$cnt2} eq "+") and ($START4{$cnt2} > $START5{$cnt2})){
                my $str5=substr($seq1,($START5{$cnt2}-1),($END5{$cnt2}-$START5{$cnt2}+1)).substr($seq1,($START4{$cnt2}-1),($END4{$cnt2}-$START4{$cnt2}+1));
                print $out_coding ">".$GENE2{$cnt2}."_".$SP2{$cnt2}."\n".$str5."\n";
            }
        }elsif ((defined $STRAND5{$cnt2} ne "") and (defined $STRAND6{$cnt2} ne "")) {
            if (($STRAND4{$cnt2} eq "-") and ($START4{$cnt2} < $START5{$cnt2}) and ($START5{$cnt2} < $START6{$cnt2})) {
                my $str6=substr($seq1,($START4{$cnt2}-1),($END4{$cnt2}-$START4{$cnt2}+1)).substr($seq1,($START5{$cnt2}-1),($END5{$cnt2}-$START5{$cnt2}+1)).substr($seq1,($START6{$cnt2}-1),($END6{$cnt2}-$START6{$cnt2}+1));
                my $rev_com4=reverse $str6;
                $rev_com4=~ tr/ACGTacgt/TGCAtgca/;
                print $out_coding ">".$GENE2{$cnt2}."_".$SP2{$cnt2}."\n".$rev_com4."\n";
            }elsif(($STRAND4{$cnt2} eq "-") and ($START4{$cnt2} > $START5{$cnt2}) and ($START5{$cnt2} > $START6{$cnt2})) {
                my $str7=substr($seq1,($START6{$cnt2}-1),($END6{$cnt2}-$START6{$cnt2}+1)).substr($seq1,($START5{$cnt2}-1),($END5{$cnt2}-$START5{$cnt2}+1)).substr($seq1,($START4{$cnt2}-1),($END4{$cnt2}-$START4{$cnt2}+1));
                my $rev_com5=reverse $str7;
                $rev_com5=~ tr/ACGTacgt/TGCAtgca/;
                print $out_coding ">".$GENE2{$cnt2}."_".$SP2{$cnt2}."\n".$rev_com5."\n";
            }elsif(($STRAND4{$cnt2} eq "-") and ($START4{$cnt2} > $START6{$cnt2}) and ($START6{$cnt2} > $START5{$cnt2})) {
                my $str8=substr($seq1,($START4{$cnt2}-1),($END4{$cnt2}-$START4{$cnt2}+1));
                my $str9=substr($seq1,($START5{$cnt2}-1),($END5{$cnt2}-$START5{$cnt2}+1)).substr($seq1,($START6{$cnt2}-1),($END6{$cnt2}-$START6{$cnt2}+1));
                my $rev_com6=reverse $str8;
                $rev_com6=~ tr/ACGTacgt/TGCAtgca/;
                print $out_coding ">".$GENE2{$cnt2}."_".$SP2{$cnt2}."\n".$rev_com6.$str9."\n";
            }elsif(($STRAND4{$cnt2} eq "+") and ($START4{$cnt2} < $START5{$cnt2}) and ($START5{$cnt2} < $START6{$cnt2})){
                my $str10=substr($seq1,($START4{$cnt2}-1),($END4{$cnt2}-$START4{$cnt2}+1)).substr($seq1,($START5{$cnt2}-1),($END5{$cnt2}-$START5{$cnt2}+1)).substr($seq1,($START6{$cnt2}-1),($END6{$cnt2}-$START6{$cnt2}+1));
                print $out_coding ">".$GENE2{$cnt2}."_".$SP2{$cnt2}."\n".$str10."\n";
            }elsif(($STRAND4{$cnt2} eq "+") and ($START4{$cnt2} > $START5{$cnt2}) and ($START5{$cnt2} > $START6{$cnt2})){
                my $str11=substr($seq1,($START6{$cnt2}-1),($END6{$cnt2}-$START6{$cnt2}+1)).substr($seq1,($START5{$cnt2}-1),($END5{$cnt2}-$START5{$cnt2}+1)).substr($seq1,($START4{$cnt2}-1),($END4{$cnt2}-$START4{$cnt2}+1));
                print $out_coding ">".$GENE2{$cnt2}."_".$SP2{$cnt2}."\n".$str11."\n";
            }elsif(($STRAND4{$cnt2} eq "+") and ($START4{$cnt2} > $START6{$cnt2}) and ($START6{$cnt2} > $START5{$cnt2})) {
                my $str12=substr($seq1,($START4{$cnt2}-1),($END4{$cnt2}-$START4{$cnt2}+1)).substr($seq1,($START5{$cnt2}-1),($END5{$cnt2}-$START5{$cnt2}+1)).substr($seq1,($START6{$cnt2}-1),($END6{$cnt2}-$START6{$cnt2}+1));
                print $out_coding ">".$GENE2{$cnt2}."_".$SP2{$cnt2}."\n".$str12."\n";
            }
        }
    }
    close $out_coding;
    @output1=();


    #generate_IGS_ranges
    my (%SP3,%GENE3,%STRAND7,%START7,%END7,%TYPE7,$last0,$last1,$last2,@output4);
    my $cnt3=0;
    foreach (@output3){
        chomp;
        $cnt3++;
        ($SP3{$cnt3},$GENE3{$cnt3},$STRAND7{$cnt3},$START7{$cnt3},$END7{$cnt3},$TYPE7{$cnt3})=(split /\s+/,$_)[0,1,2,3,4,5];
    }
    foreach (keys %SP3){
        if ($_==1 and $START7{$_}!=1){
            unshift @output4,$SP3{$_}."\t"."start".'-'.$GENE3{$_}."\t"."?"."/".$STRAND7{$_}."\t"."1"."\t".($START7{$_}-1)."\t"."?"."/".$TYPE7{$_}."\n";
        }
    }
    foreach (1..($cnt3-1)) {
        $last0=$_-1;
        $last1=$_+1;
        $last2=$_+2;
        next if ((($END7{$_}+1) >= ($START7{$last1}-1)) and (($END7{$_}+1) < ($END7{$last1}-1)));
        next if (($_ > 1) and (($END7{$_}+1) < ($END7{$last0}-1)) and (($END7{$_}+1) < ($START7{$last2}-1)));
        if ((($END7{$_}+1) >= ($START7{$last1}-1)) and (($END7{$_}+1) >= ($END7{$last1}-1))){
            push @output4,$SP3{$_}."\t".$GENE3{$_}.'-'.$GENE3{$last2}."\t".$STRAND7{$_}."/".$STRAND7{$last2}."\t".($END7{$_}+1)."\t".($START7{$last2}-1)."\t".$TYPE7{$_}."/".$TYPE7{$last2}."\n";
        }else{
            push @output4,$SP3{$_}."\t".$GENE3{$_}.'-'.$GENE3{$last1}."\t".$STRAND7{$_}."/".$STRAND7{$last1}."\t".($END7{$_}+1)."\t".($START7{$last1}-1)."\t".$TYPE7{$_}."/".$TYPE7{$last1}."\n";
        }
    }
    foreach (keys %SP3){
        if ($_==$cnt3){
            push @output4,$SP3{$_}."\t".$GENE3{$_}.'-'."end"."\t".$STRAND7{$_}."/"."?"."\t".($END7{$_}+1)."\t".$length."\t".$TYPE7{$_}."/"."?"."\n";
        }
    }
    @output3=();


    #extract_IGS
    my (%SP4,%GENE4,%STRAND8,%START8,%END8,%TYPE8,$seq2);
    my $cnt4=0;
    open(my $out_noncoding,">","$filename_base\_intergenic.fasta");
    while (@fasta2){
        my $header=shift @fasta2;
        $seq2=shift @fasta2;
    }
    foreach (@output4){
        chomp;
        $cnt4++;
        ($SP4{$cnt4},$GENE4{$cnt4},$STRAND8{$cnt4},$START8{$cnt4},$END8{$cnt4},$TYPE8{$cnt4})=(split /\s+/,$_)[0,1,2,3,4,5];
        my $str=substr($seq2,($START8{$cnt4}-1),($END8{$cnt4}-$START8{$cnt4}+1));
        print $out_noncoding ">".$GENE4{$cnt4}."_".$SP4{$cnt4}."\n".$str."\n";
    }
    @output4=();
    close $out_noncoding;
    unlink "$filename_base\_intergenic.fasta";
}