Hello everyone

I'm trying to design primers for microsatellite markers. I used Auto_primer_v1.0 (on CentOS) which included TRF to find SSR and Primer3 to design the primers. However, whenever i run auto_primer with the following command and parameters, the result showed potential repeat motifs but no primers found. I was thinking that because the motifs are generally next to each other so Primer3 couldn't find the flanking region (Short Flanking). The other error was full sequence is too short (< 100 bp).

$ perl auto_primer1.0.pl FINAL.fa Final -min 2 -max 8 -min_product 100 -max_product 400 -noqual

I've also tried to modify the Tm range (minTM: 54, maxTM: 68), production range (from max: 400 to 1000 bp), min flanking (from 30 down to 10 and up to 50) but none of those seemed to work.

The input (FINAL.fa) is genome fasta file from Illumina.

So can anyone tell me where is the problem ? And possibly how to solve it ?

Thank you in advance