# pcr.seqs start/stop for V3\_5 on MiSeq **URL:** \ **Category:** Theory behind mothur\ **Created:** [December 6, 2013, 9:29pm UTC](https://forum.mothur.org/t/pcr-seqs-start-stop-for-v3-5-on-miseq/1608 "2013-12-06T21:29:03Z")\ **Posts on this page:** 8\ **Page:** 1
**Author:** ![klgreathouse](https://avatars.discourse-cdn.com/v4/letter/k/94ad74/32.png) [@klgreathouse](https://forum.mothur.org/u/klgreathouse)\ **Post date:** [December 6, 2013, 9:29pm UTC](https://forum.mothur.org/t/pcr-seqs-start-stop-for-v3-5-on-miseq/1608/1 "2013-12-06T21:29:03Z")
HI, 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? Thanks, Leigh ---
**Author:** ![pschloss](https://yyz2.discourse-cdn.com/flex036/user_avatar/forum.mothur.org/pschloss/32/4_2.png) [@pschloss](https://forum.mothur.org/u/pschloss)\ **Post date:** [December 9, 2013, 5:05pm UTC](https://forum.mothur.org/t/pcr-seqs-start-stop-for-v3-5-on-miseq/1608/2 "2013-12-09T17:05:07Z")
Try start=6426 and end=27654 Pat ---
**Author:** ![jopollock](https://avatars.discourse-cdn.com/v4/letter/j/74df32/32.png) [@jopollock](https://forum.mothur.org/u/jopollock)\ **Post date:** [December 27, 2013, 3:18pm UTC](https://forum.mothur.org/t/pcr-seqs-start-stop-for-v3-5-on-miseq/1608/3 "2013-12-27T15:18:23Z")
Hello, 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, Jo ---
**Author:** ![pschloss](https://yyz2.discourse-cdn.com/flex036/user_avatar/forum.mothur.org/pschloss/32/4_2.png) [@pschloss](https://forum.mothur.org/u/pschloss)\ **Post date:** [January 6, 2014, 8:30pm UTC](https://forum.mothur.org/t/pcr-seqs-start-stop-for-v3-5-on-miseq/1608/4 "2014-01-06T20:30:44Z")
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. ---
**Author:** ![klgreathouse](https://avatars.discourse-cdn.com/v4/letter/k/94ad74/32.png) [@klgreathouse](https://forum.mothur.org/u/klgreathouse)\ **Post date:** [January 14, 2014, 3:19pm UTC](https://forum.mothur.org/t/pcr-seqs-start-stop-for-v3-5-on-miseq/1608/5 "2014-01-14T15:19:59Z")
Thanks, that’s what I was also able to find from alignment to e.coli! 😃 ---
**Author:** ![FM\_Kerckhof](https://yyz2.discourse-cdn.com/flex036/user_avatar/forum.mothur.org/fm_kerckhof/32/32_2.png) [@FM\_Kerckhof](https://forum.mothur.org/u/FM_Kerckhof)\ **Post date:** [September 4, 2014, 11:52pm UTC](https://forum.mothur.org/t/pcr-seqs-start-stop-for-v3-5-on-miseq/1608/6 "2014-09-04T23:52:06Z")
Hi, 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: forward CCTACGGGNGGCWGCAG (341F) reverse GACTACHVGGGTATCTAAKCC (785R) Just running pcr.seqs with these primers returns an empty fasta… which worries me a bit. Am I overlooking some things here? edit 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, FM ---
**Author:** ![pschloss](https://yyz2.discourse-cdn.com/flex036/user_avatar/forum.mothur.org/pschloss/32/4_2.png) [@pschloss](https://forum.mothur.org/u/pschloss)\ **Post date:** [September 10, 2014, 5:28pm UTC](https://forum.mothur.org/t/pcr-seqs-start-stop-for-v3-5-on-miseq/1608/7 "2014-09-10T17:28:34Z")
I would take the sequence at [http://rdp.cme.msu.edu/hierarchy/detail.jsp?seqid=S000000258&format=fasta](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. ---
**Author:** ![FM\_Kerckhof](https://yyz2.discourse-cdn.com/flex036/user_avatar/forum.mothur.org/fm_kerckhof/32/32_2.png) [@FM\_Kerckhof](https://forum.mothur.org/u/FM_Kerckhof)\ **Post date:** [February 19, 2015, 3:57pm UTC](https://forum.mothur.org/t/pcr-seqs-start-stop-for-v3-5-on-miseq/1608/8 "2015-02-19T15:57:07Z")
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, FM