pcr.seqs start/stop for V3_V4 on MiSeq


I am trying to determine what the “start” and “end” parameters should be for V3_4 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?



Why not trying with the option oligo which would include a file that has your primers?

But anyway, V3_V4 is too long to have good overlapping sequences in your final contigs assembly so I do not think that Mothur is the tool you should use. you may run into problems later on.

If you have a mock community as a positive control, that will help you greatly also.

Hope it helps!

Hi Alex,

What tool other than mothur would you recommend for V3-V4 analysis?


I wouldn’t recommend anything but mothur. I’d recommend not sequencing V3-V4.



Cannot argue against the Creator!
There is a couple of things I always wanted to test (just for fun because well I like testing stuff)
But if you are stuck with what you have, you might want to try Qiime (never tried it)
Or use trimmomatic to trim your fastq files based on quality to create other fastq files to use with Mothur to try to reduce your error rate before starting Mothur (never tried it).
Ideally with a mock community so you have a basis for comparing your methodologies.
That may improve output, but long sequences, not overlapping = lots of nucléotides without error correction = lots of errors that eventually are becoming so common they are treated as a “real” population and are hard to detect and remove. You still will have to take that into account when writing your paper and to compare with the existing littérature.
Good luck!

Hi all,
Thanks for the replies. I was not involved in the the sequencing of the data, I was just given it to analyse. I’ll talk to my PI about the possibility of re-sequencing the samples using just the v4 section. Once again, thanks for the replies.

Hi, maybe a bit too late to the party but a very straightforward way to determine these positions is to just pick an (any) E. coli sequence (e.g. X80725) use a text editor to match your primer sequences against it (make sure to reverse complement your reverse primer) and remove anything outside of the primer region. Then, align this E. coli to the database of your choice (e.g. the recreated SILVA seed). Look at summary.seqs and you have it: the values for your start and stop (at least via coli numbering). We use a modified version of the V3/V4 primers from Klindworth et al (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3592464/) 341F/785R and for us the positions are 6388 and 25316 respectively.

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I also wrote a tutorial on finding the coordinates here…