pcr.seqs with silva.seed_v119

Commands in mothur
1

Hi,

I am trying to create a smaller reference alignment to use with MiSeq data.
I am using primers targeting the ecoli 27F-519R region.
I include these in the oligos file:

forward AGRGTTTGATCMTGGCTCAG
reverse GTNTTACNGCGGCKGCTG


But when i run the pcr.seqs command:

pcr.seqs(fasta=silva.seed_v19.fasta, oligos=primers.txt, keepprimer=true, keepdots=false, pdiffs=3)


all of the sequences except 1 end up in the scrap file. Ive tried changing the value for pdiffs but with no real improvement.

If anyone has any ideas id really appreciate them

Kind regards

Guy Abell…

2

I suspect it’s because none of the sequences have the 27f primer. That primer has been trimmed off at position 1044 because hardly anyone (except those doing genome sequences) have actually sequenced the 27f primer.

Pat

3

Ah, ok that explains it then.
Thanks Pat!

Guy