Find simple repeat sequences in the assembly genome and design proper primers for simple repeat sequences in 10 steps ssr_finder
Find simple repeat sequences in the assembly genome and design proper primers for simple repeat sequences
####################To get ssr from the genome################### 1ssr_finder: to get ssr,find the tandem repeat from the target sequences ,you can define ssr repeat numbers
and the flank length of ssr yourself or you can use default (motif two >= six times, motif three, motif four, motif five, motif six >= four times, flank length of ssr is 150bp)
2primer_designer: design ssr primer
3filter_primer: filter the primers which have ssr(2-6 basepair,repeats>=4)
4： formatdb:the genome file which need analyse and blast the primers to the genome, blast the primer sequence up to the target file, prevent the primer file multi-mapping to the target region. #####sometimes it need to use parameter -b 10000 -v 10000###########
5blast_parse: get the primer which 5' of the primer have 3 mismatch and 3' of primer have 1 mismatch
6generate_primer_pair_new: get the primer that match to the position, extract the pair primers which map to the same scaffold
7final_pair: get the intermediate primer, the product of the pair primer is larger than 1000, you can use the default value or you can define yourself.
8product_ssr_check:get the product by intermediate primer from the given genome
ssr_finder:check ssr in product, repeat numbers of motif should be the same with 1ssr_finder.pl
9filter_ssr: filter the product that contain more than one ssr,one pair primer one ssr
10get_fitprimer: get the suitable primer and ssr(sort by repeat units and repeat numbers) ################################################################## The format in 10get_fitprimer.pl is follow array1: scaffold ID
array2: ssr sequence
array3: ssr units(ssr units length * copys)
array4: the position of the ssr on the scaffold
array5: length of ssr
array6: product of ssr------------ssr_left[ssr]ssr_right
array7: sequences of the forward primer
array8: the suitable amplification temperature for the forward primer
array9: sequences of the reverse primer
array10: the suitable amplification temperature for the reverse primer
array11: length of the amplification product
############################## This procedure is to get all ssr from genome. After this process, you can define how many ssrs on one scaffold (ssr per scaffold), so you need to think carefully;
#################################################################### step4 is time-consuming, maybe >=120min, but memory is low,you can run on login nod or you can qsub ####################################################################
#################path is the directory contained perl scripts#################### perl $path/1ssr_finder --flank 150 --ssr2 6 --ssr3 4 --ssr4 4 --ssr5 4 --ssr6 4 $1 $outdir/$2.ssr
perl $path/2primer_designer $outdir/$2.ssr $outdir/$2.raw_primer $outdir/$2.primer_result
perl $path/3filter_primer $outdir/$2.primer_result $outdir/$2.rescreen $outdir/$2.blastin
formatdb -i $1 -p F
blastall -i $outdir/$2.blastin -d $1 -p blastn -o $outdir/$2.blast.out -F F -b 10000 -v 10000
perl $path/5blast_parse_change $outdir/$2.blast.out $outdir/$2.query_sbjct 4
perl $path/6generate_primer_pair_new $outdir/$2.query_sbjct $outdir/$2.primer.tab
perl $path/7final_pair $outdir/$2.primer.tab $outdir/$2.rescreen $outdir/$2.inter_primer.tab 2000
perl $path/8product_ssr_check $1 $outdir/$2.inter_primer.tab $outdir/$2.product_file
perl $path/1ssr_finder --ssr2 6 --ssr3 4 --ssr4 4 --ssr5 4 --ssr6 4 $outdir/$2.product_file $outdir/$2.product_ssr
perl $path/9filter_ssr $outdir/$2.product_ssr $outdir/$2.inter_primer.tab $outdir/$2.only_primer.tab
perl $path/10get_fitprimer $outdir/$2.only_primer.tab $outdir/$2.product_ssr $outdir/$2.rescreen $outdir/$2.final_primer
Warning: Maybe you need to install perl package "ByteLoader"(http://search.cpan.org/~rurban/B-C-1.55/ByteLoader/ByteLoader.pm) to run this pipeline, this pipeline was tested under perl5.10.