在提取miRNA前后500bp序列时，若没有其位置信息，提取会比较麻烦，可用以下方法提取：

1.首先利用blast软件将miRNA的前体序列与基因组比对，获取前体序列的位置信息，比对方法：

makeblastdb -in Donkey_Hic_genome.20180408.fa -dbtype nucl -title Donkey_Hic_genome.20180408.fa
blastall -i miRNA_Pre.fa -d Donkey_Hic_genome.20180408.fa -p blastn -e 0.01 -b 5 -v 5 -m 8 -o blast.out
#Donkey_Hic_genome.20180408.fa  参考基因组染色体序列
#miRNA_Pre.fa  miRNA的前体序列

由于序列可能较短，所以e-value值可调高一些。比对结果如下：

bta-miR-34c     Chr20   100.00  113     0       0       1       113     39385825        39385713        6e-57    224
bta-miR-370     Chr07   100.00  109     0       0       1       109     70327160        70327268        1e-54    216
bta-miR-34b     Chr20   100.00  112     0       0       1       112     39386420        39386309        2e-56    222
bta-miR-383     Chr27   100.00  110     0       0       1       110     21958895        21959004        4e-55    218
bta-miR-449a    Chr01   100.00  112     0       0       1       112     103603665       103603554       2e-56    222
bta-miR-378     Chr09   100.00  111     0       0       1       111     51049164        51049054        9e-56    220
bta-miR-378     Chr13   96.88   32      1       0       67      98      32631593        32631624        3e-06   56.0
bta-miR-378     Chr13   93.94   33      2       0       62      94      29900614        29900582        2e-04   50.1
bta-miR-378     Chr07   100.00  27      0       0       71      97      65072541        65072515        1e-05   54.0
bta-miR-378     Chr30   96.55   29      1       0       69      97      28926810        28926782        2e-04   50.1
bta-miR-378     Chr18   93.94   33      2       0       62      94      21721834        21721866        2e-04   50.1
bta-miR-206     Chr08   100.00  112     0       0       1       112     78794660        78794771        2e-56    222
bta-miR-1       Chr15   100.00  108     0       0       1       108     48711439        48711546        5e-54    214
bta-miR-1       Chr07   88.89   63      7       0       32      94      26712206        26712268        2e-10   69.9
bta-let-7c      Chr18   100.00  112     0       0       1       112     32412721        32412610        2e-56    222
bta-let-7c      Chr20   89.19   74      8       0       18      91      29703021        29703094        1e-14   83.8

bta-miR-34c     Chr20   100.00  113     0       0       1       113     39385825        39385713        6e-57    224
bta-miR-370     Chr07   100.00  109     0       0       1       109     70327160        70327268        1e-54    216
bta-miR-34b     Chr20   100.00  112     0       0       1       112     39386420        39386309        2e-56    222
bta-miR-383     Chr27   100.00  110     0       0       1       110     21958895        21959004        4e-55    218
bta-miR-449a    Chr01   100.00  112     0       0       1       112     103603665       103603554       2e-56    222
bta-miR-378     Chr09   100.00  111     0       0       1       111     51049164        51049054        9e-56    220
bta-miR-206     Chr08   100.00  112     0       0       1       112     78794660        78794771        2e-56    222
bta-miR-1       Chr15   100.00  108     0       0       1       108     48711439        48711546        5e-54    214
bta-let-7c      Chr18   100.00  112     0       0       1       112     32412721        32412610        2e-56    222

perl /share/work/wangq/script/lv/miRNA_500_fasta.pl -id blast.out  -fa Donkey_Hic_genome.20180408.fa  -out result.fa  -miR miRNA.fa -l 500

-id 后跟筛选的blast结果；-fa后跟参考基因组染色体序列；-miR后跟miRNA的序列文件；-l后跟提取miRNA前后多长序列，默认500。

提取结果（miRNA序列大写标注，其余小写）：

>bta-miR-34c
acaaggcacagcatcacccgccgccctgctgggaggaggccgccaccctccgcggcgaactgccgacccgaagcgtttgagaggagaaagctgcgcttcgagactggatgcgtcagcatttcttccgcgcggcgcggcgagcgcgtggtccgcgtgcgagcaaaccccctgcaaacgcaggcgggctgatgcttctagctggagttaaacagttagctatcactaggagtaaaagcaagattgggcgatcccatgtaaggaaagcaaaatctggggcgtttagtagctttaattgtaaggtgtaaagacatttgaattgctgggggaagccccctgtgtaacgttcagagctgtgagtcactgtgcctattttccattgtttatcagggtactcaccaatccaccaactaaagtgagtaccacggagccagtgatctgcctgtcacgacgcatgggggcaccaacttgagactgaagtttgtgatgaaagtaaagcttttttgctgtgagtctagttactAGGCAGTGTAGTTAGCTGATTGctaataataccaatcactaaccacacggccaggtaaaaagatttgggaattcatccagatgagctgcgtgtgcacaccagtgggtttgggggcaagaaggggattggaataccctaatagtacgcattgcctgtttatccatagctcagccaagagagaaatcagcattttagctgctaaatatacaacatatgtagtaaatatacatttttaacatataatttttcaatattccttcaggcgcttaaccaaaaaaattttcagatatattgaggaatagaggttttctctcttagctcatttatgtcattgtgtaaacttgtgattattttgaactatctgtaagactgtattactattttgaaagaataaagtgccctaaagtcataaattgtggtcattcatgtgtccattgcctttcctaagttggctttatgatgtccttctcagcgcctgccacagtaattataactttcctatcgctaaaatggttaaattgttcctagattaacaaaatctcccgggaaggctattcacaagcactttttagtatttttctaagaacacgtta
>bta-miR-370
tgtttattgttcgtgtctcagagttgctctgaggccagtgtgctgggcacccagcaggccatctgggaggggagaaaggaagctagccatgtatgtcagtaggggtcagcgtggaggcctttggggtttctgggaaacggttcgaacatggagaatctcgtgatgtggacgcccccgagggcagccccatttcatgaactggatcccttagtcggatttctgttttccagggcacttgtttaagctttcatgccgtgtccaaaaaaaagagcagatcccaagagtttcctgcccgaggcccgtcttgccagaagccctcggcttggcctgagcatcgagatcattcctctaatcagcctgggggtggtctgacccgcggggtgggcggcgtggggcggggaggggcagggcgtgtgcggggcgggagctgctgggggagctggcggcggttcctttcaccctcgccgtggacccgcgggggcgtggctgtcctcggtctacaaatcgcgcaagtcggggcacaagacagagaggccaggtcacgtctctgcagttacacagctcatgagtGCCTGCTGGGGTGGAACCTGGTctgtctgtctgtctagcaccacagctcgggcgctgctgcagagggaacaaagatttgggtgagggcctcagagacgggctggggaaggggtttactcgggctgactttgacatgaaaacaatagctaattctgcttggggcctagtgctgtgactatcttacagatgggaaacaggcacagacaggttagttaactttcctgatgctttccagctcatcaggattggaggctggatttggaggcaggcggtgtggttcgagcatatgtgctctaaccattgggaaacactgcctcctgactgtgagcacacgtagagatggcacatggagtccaagaatctgggtttgagtccttttaccactgcctggtgggtgtactgtccagtcaatcatacatatttcattcttcctcttgacagagtctctgcagaggcccagcgagtcgttggcccgtgggaaatatttttttaagaatgcatgtgtgtgatagaattttatatctatcgaaggggagggg

脚本代码如下：

#!/usr/bin/perl -w
use strict;
use Getopt::Long;
use Bio::SeqIO;
use Bio::Seq;
my $version = "1.3";
my %opts;
GetOptions(\%opts,  "id=s", "fa=s", "miR=s", "l=s","out=s","h");
if(!defined($opts{out}) || !defined($opts{fa}) || !defined($opts{miR}) || !defined($opts{id}) ||defined($opts{h}))
{
print <<"Usage End.";
Description:
$version:lefse analysis
Usage
Forced parameter:
-id           blast.out                    <infile>     must be given
-out          outfile                        <outfile>        must be given
-fa      genome fasta file    <infile>must be given
                -miR          miRNA fasta file              <infile>must be given
                -l            length                        <int>        
Other parameter:
-h           Help document
Usage End.
exit;
}
my $len = $opts{l};
$len ||=500;
my $in  = Bio::SeqIO->new(-file => "$opts{fa}" , -format => 'Fasta');
my %fasta;
while ( my $seq = $in->next_seq() ) {
my($id,$sequence)=($seq->id,$seq->seq);
$fasta{$id}=$sequence;
}

my $ina  = Bio::SeqIO->new(-file => "$opts{miR}" , -format => 'Fasta');
my %mi;
while ( my $seq = $ina->next_seq() ) {
        my($id,$sequence)=($seq->id,$seq->seq);
        $mi{$id}=$sequence;
}

open(IN,"$opts{id}") ||die "open file $opts{id} faild.\n";
open(OUT,">$opts{out}") ||die "open file $opts{out} faild.\n";
while(<IN>){
chomp;
my @line = split("\t");
if($line[8] < $line[9]){
my $miR = lc(substr( $fasta{$line[1]},$line[8]-1, $line[9] - $line[8]+1));
my $small = lc($mi{$line[0]});
$miR =~ s/$small/$mi{$line[0]}/;
my $before = lc(substr( $fasta{$line[1]},$line[8]-$len-1, $len));
my $laft = lc(substr( $fasta{$line[1]},$line[9], $len));
print OUT ">$line[0]\n$before$miR$laft\n";
}elsif($line[8] > $line[9]){
my $miR = lc(substr( $fasta{$line[1]},$line[9]-1, $line[8] - $line[9]+1));
my $before = lc(substr( $fasta{$line[1]},$line[9]-$len-1, $len));
my $laft = lc(substr( $fasta{$line[1]},$line[8], $len));
my $gene = $before.$miR.$laft;
$gene = &reverse_complement_IUPAC($gene);
my $small = lc($mi{$line[0]});
$gene =~ s/$small/$mi{$line[0]}/;
print OUT ">$line[0]\n$gene\n";
}
}
close(IN);
close(OUT);
sub reverse_complement_IUPAC {
        my $dna = shift;
        # reverse the DNA sequence
        my $revcomp = reverse($dna);
        # complement the reversed DNA sequence
        $revcomp =~ tr/ABCDGHMNRSTUVWXYabcdghmnrstuvwxy/TVGHCDKNYSAABWXRtvghcdknysaabwxr/;
        return $revcomp;
}