Primer.design

Commands in mothur
1

Hello Mothur group,

I would like to know how can i design a primer for one specific bacteria of my interest. I tried using mothur in silico command for this but it generated lot of primers and it was OTU wise and generated more primers than total number of OTU’s.

here is the command i used.
primer.design(list=stability.trim.contigs.good.unique.good.filter.unique.precluster.pick.pick.subsample.an.list, fasta=stability.trim.contigs.good.unique.good.filter.unique.precluster.pick.pick.subsample.fasta,name=stability.trim.contigs.good.unique.good.filter.unique.precluster.pick.pick.subsample.names,otunumber=5)

I might be interpreting or doing it wrong. Please suggest how can it be seen.

Thanks

2

You might try throwing on label=unique. After that you’ll have to do some troubleshooting for things like length, Tm, hairpins, etc.

Pat

3

Thank for reply Pat. I agree troubleshooting is required. I did the way you suggested (but using label=0.03) and now its fine.
but there are some things which i would like to know

primer.design(list=stability.trim.contigs.good.unique.good.filter.unique.precluster.pick.pick.subsample.an.list, fasta=stability.trim.contigs.good.unique.good.filter.unique.precluster.pick.pick.subsample.fasta, name=stability.trim.contigs.good.unique.good.filter.unique.precluster.pick.pick.subsample.names, otunumber=5,label=0.03,processors=16)

The output provides primers for all otu’s apart from number mentioned in command is this normal output?

Thanks

4

The primer design command finds the consensus sequences for each OTU. It then finds the primers for the OTU you selected, in your case number 5. Mothur will then search each of the other OTUs consensus sequences for the primers found in the selected OTU (#5) and report the results to you.