conda install -c bioconda last

    lastdb -P8 -uMAM8 myDB $ref
    
    ##first query
    
    last-train -P40 --revsym -E0.05 -C2 myDB $query1 >$sp1.train
    lastal -k32 -p $sp1.train myDB $query1 | last-split -fMAF+ >$sp1.maf
    maf-swap $sp1.maf |last-split |maf-swap |maf-sort > $sp1.filter.maf
    
    ##second query
    
    last-train -P40 --revsym -E0.05 -C2 myDB $query2 >$sp2.train
    lastal -k32 -p $sp2.train myDB $query2 | last-split -fMAF+ >$sp2.maf
    maf-swap $sp2.maf |last-split |maf-swap |maf-sort > $sp2.filter.maf

    perl maf.rename.species.S.pl $sp1.filter.maf $sp1 $sp1.last.maf >$sp1.rename.maf
    perl maf.rename.species.S.pl $sp2.filter.maf $sp2 $sp2.last.maf >$sp2.rename.maf

    ## multiz合并maf(>3个query according to phylogenetic tree for merging)
    multiz M=1 $sp1.rename.maf $sp2.rename.maf 0 U1 U2 > $sp1-$sp2.ma        

    perl 01.convertMaf2List.pl $sp1-$sp2.maf $sp1-$sp2 $sp_ref $sp1,$sp2

    perl 02.lst2gene.pl $sp1-$sp2 $gff

    perl filter.pl [fasta] [filter_file]

    for i in genes/*.filter; do echo -e "iqtree -s $i" >> iqtree.sh; done
    for i in genes/*.treefile; do echo -e "nw_reroot  $i  imhausi > $i.nwk" >> reroot.sh; done
    for i in genes/*.nwk; do echo -e "hyphy label-tree.bf --tree $i --list chr.list --output $i.label" >> label.sh ;done

    hyphy absrel --alignment genes/evm.model.Contig11.1.fa.filter \
    --tree genes/evm.model.Contig11.1.fa.filter.treefile.tree.nwk \
    --branches Foreground \
    -output genes/evvm.model.Contig11.1.fa.filter.result

    hyphy busted --alignment genes/evm.model.Contig11.1.fa.filter \
    --tree genes/evm.model.Contig11.1.fa.filter.treefile.tree.nwk \
    --branches Foreground \
    -output genes/evvm.model.Contig11.1.fa.filter.result

    hyphy relax --alignment  genes/evm.model.Contig11.1.fa.filter \
    --test Foreground \
    --tree genes/evm.model.Contig11.1.fa.filter.treefile.tree.nwk \
    --output genes/evm.model.Contig11.1.fa.filter.relax