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primer3-org / primer3

Primer3 is a command line tool to select primers for polymerase chain reaction (PCR).
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Primer3 not finding microsatellite primers from reference genome #37

Closed ctdarwell closed 2 years ago

ctdarwell commented 4 years ago

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 (I used my own script to include 150bp either side of the identified SSR) 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 please explain 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 downloaded 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?

If P3 cannot find particularly likely candidate primers, what strategy should I use to select the best potential primers from the files I have created?

Any help much appreciated thanks

Clive

untergasser commented 2 years ago

Hi, can you send me the input file for Primer3? Best, Andreas