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In Silico Pcr - Small Coverage

I have a set of primers designed by somebody else on the basis of Arb Silva SSU database (specifically, all are matching different fragments of 16S rRNA). The issue I have is that I cannot reproduce the depth of the coverage these primers should have according to the papers describing them with anything else than TestPrime server, Arb Silva's own software. I've tried e-PCR and MFEPrimer2.0, both with quite liberal conditions. I attempted to catch the sequences these primers _have_ matched in the real world PCR (I have amplicons already sequenced) and still I don't get enough coverage (like 5-10% of the things that are there). Is there anything I'm missing here?

pcr
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updated by Biostar Biostar • written by Pawel Szczesny Pawel Szczesny

Perhaps you could blast the primers against the known targets and see whether they are there at all in the first place, then check for correct orientation/pairing - just eliminating some possible problems. I am kind of thinking out loud.

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written by Istvan Albert Istvan Albert

Thanks for the suggestion. Blast (adjusted for short sequences) shows correct number of sequences matching the primers. I have tested all possible versions of primers (reversed, complement) I am sure I have the correct orientation and pairing.

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written by Pawel Szczesny Pawel Szczesny

1 answer

The problem is solved, although not in a way I've hoped for. I've tried the third software, ipcress (part of exonerate package from Guy Slater at EBI; it's in the repositories of all major linux distribution). It gives correct results under liberal conditions (certain number of mismatches allowed). Still I have no idea why two other programs failed to give comparable results.

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written by Pawel Szczesny Pawel Szczesny

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