# pcr.seqs **URL:** \ **Category:** Commands in mothur\ **Created:** [July 25, 2014, 3:23pm UTC](https://forum.mothur.org/t/pcr-seqs/1940 "2014-07-25T15:23:46Z")\ **Posts on this page:** 12\ **Page:** 1
**Author:** ![dennerdr](https://avatars.discourse-cdn.com/v4/letter/d/a587f6/32.png) [@dennerdr](https://forum.mothur.org/u/dennerdr)\ **Post date:** [July 25, 2014, 3:23pm UTC](https://forum.mothur.org/t/pcr-seqs/1940/1 "2014-07-25T15:23:46Z")
Hi. I am brand new to mothur (taking the workshop in August) and brand new to a project in my lab that has been sitting and waiting for someone to pick up. Thus…I have a very general question. Is there an easy, straight-forward way of determining the start and end point for my sequences of interest? Thanks ---
**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:** [July 25, 2014, 5:23pm UTC](https://forum.mothur.org/t/pcr-seqs/1940/2 "2014-07-25T17:23:21Z")
Looking forward to meeting you! Try this… 1. Take ecoli’s 16s and trim it to the region being amplified by your primers 2. Align the trimmed sequence to silva.bacteria.fasta 3. Run the \*align file through summary.seqs - take note of the start and end positions ---
**Author:** ![dennerdr](https://avatars.discourse-cdn.com/v4/letter/d/a587f6/32.png) [@dennerdr](https://forum.mothur.org/u/dennerdr)\ **Post date:** [July 28, 2014, 8:36pm UTC](https://forum.mothur.org/t/pcr-seqs/1940/3 "2014-07-28T20:36:19Z")
THANKS! Unfortunately though I’ve run into another problem. Now when I run my pcr.seqs command everything looks like it’s going fine until its gets about a fourth in and I get these errors: [ERROR]: 26590.temp is blank. Please correct. [ERROR]: 26590.temp is blank. Please correct. [ERROR]: name mismatch in pcr.seqs. [ERROR]: name mismatch in pcr.seqs. The second error (name mismatch in pcr.seqs) occurs about a million times (exaggerating a little). Thoughts??? Thanks! Looking forward to meeting you in a month as well! Darcy ---
**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:** [July 29, 2014, 7:22pm UTC](https://forum.mothur.org/t/pcr-seqs/1940/4 "2014-07-29T19:22:11Z")
Are you doing this in windows, mac, or linux? How many processors? Can you try reducing the number of processors? ---
**Author:** ![dennerdr](https://avatars.discourse-cdn.com/v4/letter/d/a587f6/32.png) [@dennerdr](https://forum.mothur.org/u/dennerdr)\ **Post date:** [July 30, 2014, 3:01pm UTC](https://forum.mothur.org/t/pcr-seqs/1940/5 "2014-07-30T15:01:10Z")
I’m doing this on a Mac with 1 processor. ---
**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:** [July 30, 2014, 4:55pm UTC](https://forum.mothur.org/t/pcr-seqs/1940/6 "2014-07-30T16:55:21Z")
What’s the exact syntax of the command you’re trying to run? ---
**Author:** ![dennerdr](https://avatars.discourse-cdn.com/v4/letter/d/a587f6/32.png) [@dennerdr](https://forum.mothur.org/u/dennerdr)\ **Post date:** [July 30, 2014, 6:30pm UTC](https://forum.mothur.org/t/pcr-seqs/1940/7 "2014-07-30T18:30:59Z")
I’ve tried it two ways: 1. with batch file as follows make.contigs(file=stability.files, processors=1) summary.seqs(fasta=stability.trim.contigs.fasta) screen.seqs(fasta=stability.trim.contigs.fasta, group=stability.contigs.groups, maxambig=0, maxlength=275) summary.seqs(fasta=current) get.current() unique.seqs(fasta=stability.trim.contigs.good.fasta) count.seqs(name=stability.trim.contigs.good.names, group=stability.contigs.good.groups) summary.seqs(count=stability.trim.contigs.good.count\_table) pcr.seqs(fasta=silva.bacteria.fasta, start=13862, end=23444, keepdots=F, processors=1) system(mv silva.bacteria.pcr.fasta silva.v4.fasta) summary.seqs(fasta=silva.v4.fasta) align.seqs(fasta=stability.trim.contigs.good.unique.fasta, reference=silva.v4.fasta) summary.seqs(fasta=stability.trim.contigs.good.unique.align, count=stability.trim.contigs.good.count\_table) get.current() 2. separately with each command as an individual ---
**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:** [July 31, 2014, 2:23pm UTC](https://forum.mothur.org/t/pcr-seqs/1940/8 "2014-07-31T14:23:56Z")
You said the command that was giving you problems was this: pcr.seqs(fasta=silva.bacteria.fasta, start=13862, end=23444, keepdots=F, processors=1) Right? I just ran that with no issue. Am I missing something? ---
**Author:** ![dennerdr](https://avatars.discourse-cdn.com/v4/letter/d/a587f6/32.png) [@dennerdr](https://forum.mothur.org/u/dennerdr)\ **Post date:** [July 31, 2014, 2:56pm UTC](https://forum.mothur.org/t/pcr-seqs/1940/9 "2014-07-31T14:56:20Z")
Right. I’m sure the command itself is fine but I’m still getting the above errors when using my data set and I don’t know where to look to attempt to fix the errors. I don’t really understand the errors themselves. Can you explain the nature of the error and perhaps I can go from there? Thanks. ---
**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:** [July 31, 2014, 6:51pm UTC](https://forum.mothur.org/t/pcr-seqs/1940/10 "2014-07-31T18:51:57Z")
I think the only way you can get those errors is if you are using multiple processors, so I’m really not sure what’s going on. What version of mothur are you using? ---
**Author:** ![cgini31](https://yyz2.discourse-cdn.com/flex036/user_avatar/forum.mothur.org/cgini31/32/429_2.png) [@cgini31](https://forum.mothur.org/u/cgini31)\ **Post date:** [December 21, 2022, 2:46pm UTC](https://forum.mothur.org/t/pcr-seqs/1940/11 "2022-12-21T14:46:08Z")
@pschloss referring to the first question of this trend, is it also a reliable method to use the coordinates you give in SOP (for V4) and [here](http://mothur.org/blog/2016/Customization-for-your-region/) (for V3) if, for example I know I have done my seqeuncing for V3 and V4 region of 16S rRNA gene? If so, is it correct if I use pcr.seqs with as parameters start=6388 and end=23444? Thanks in advance ---
**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 21, 2022, 2:57pm UTC](https://forum.mothur.org/t/pcr-seqs/1940/12 "2022-12-21T14:57:11Z")
I think that should be fine Pat