pcr.seqs start/stop for V3_5 on MiSeq


I am trying to determine what the “start” and “stop” parameters should be for V3_5 primers on MiSeq in order to run “pcr.seqs” command correctly. Is this information already available somewhere, or do I need to align my pcr product to the silva.bacteria file?


Try start=6426 and end=27654



I also have the same question, however I am focussing on the V3 region alone (primers used were 341F/518R). If this isn’t easy to find out, could someone give me instruction on how to gain this info?

Many thanks,


The best thing to do would be to take an ecoli sequence, trim it to your PCR primers, align it, and run summary.seqs and use the start/end positions.

Thanks, that’s what I was also able to find from alignment to e.coli! :smiley:


Just a quick question: I was wondering how to “trim” the E. Coli genome to my primers with mothur.
My current strategy is using a fasta of the full E. Coli K12 genome from NCBI with pcr.seqs and an oligos file containing both my forward and reverse primers:

Just running pcr.seqs with these primers returns an empty fasta… which worries me a bit.
Am I overlooking some things here?

Turns out I intially evaluated the fusion primers rather than the primers, I updated the sequences above accordingly.
Next, I was able to execute pcr.seqs without ending up with an empty fasta, but still the length of my “in silico pcr product” appears to be >3.9e+06 bases, which is of course way too long. Once again I would like to get some input on how to overcome this problem.
I am using mothur 1.33.3 on an ubuntu 12.04 server.

Kind regards,


I would take the sequence at http://rdp.cme.msu.edu/hierarchy/detail.jsp?seqid=S000000258&format=fasta and open it in something like text wrangler or notepad++ and remove the bases before your forward primer sequences and the bases after your reverse primer sequence. Save it as ecoli.fasta and align it to your reference alignment.

I didn’t reply on this yet but thanks, this really helped a lot!
For all the other users of LGC genomics’ primers 341F and 785R that may strand here: the start position for silva reference alignment v119 is 6388 and the stop position is 22096.

Kind regards,