Primer3 not finding microsatellite primers from reference genome
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17 months ago
ctdarwell • 0

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

Clive

Primer3 MISA Microsatellites SSR • 403 views
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Aren't microsatellites repettitive regions and therefore sequences are highly similar? Do surprise you get no unique primers I would say. Is this for qPCR?

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Not sure about PCR method, I was asked to find some msat primers from a published genome - identifying microsats was easy, just don't understand the output of Primer3 and find its docs pretty unclear.