Understanding pdiffs in pcr.seqs

I’m using pcr.seqs to trim my nanopore seqs using an oligos file. but I’m getting nearly all forward reads which is weird, I made my libraries with ligation so there should be roughly equal 5->3 and 3->5 reads. Which makes me think that I need to increase my pdiffs/rdiffs. How does pcr.seqs handle degeneracies in the primer? if there’s an N, will anything other than an N be counted as a mismatch by pcr.seqs or does it understand that everything matches N?

Hi Kendra,

If you put N in the primer sequence, it will match an A, T, G, or C. If the primer has an R, it will match an A or G - mothur uses the IUPAC system. If you put C in the primer sequence and the sequence has a G that will be a mismatch.


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ok then 5 mismatches seems like plenty!

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