I have a full published plant genome (280Mb). I ran MISA to identify microsatellite regions and found approx 100,000 hits. I then formatted the output files for Primer3 and ran with default settings. From this I got zero suggested primers (I increased maximum primer size and decreased minimum melting temperature as suggested - still no hits). Thus, I set PRIMER_EXPLAIN_FLAG=1 and have 200000+ (forward & reverse) files. As I understand all these discarded "primers" listed in these files failed. Could someone explain why they failed or why a complete genome would yield no hits. For example, for the following discarded primer has the following metrics:
21 20 0 50.00 59.490 3.00 1.00 0.510
Quality is 0.51 (as I understand this is good - i.e. low) - from the example file with the software the selected/outputted primers have quality of 3 (which is worse as I understand); self-annealing values are also low/good (3 & 1); GC is 50% & temp is 59.49 - are these the reasons it is not accepted under defaults?
How should I select the best potential primers from these files?
Any help much appreciated thanks