How to use Mothur software

Hi everyone
I m a new user for Mothur software.
Anyone tells me how to use Mothur software?


Look at the SOP, read the forum, try out for yourself.

Best of success

well i am trying to learn about this software.
kindly help me how to open silva.bacteria in mothur software?


this is to shallow to work with. What do you intend to do, why do you want to do ti, what is the problem you are running into?

well… when i am running pcr command it gives error i dont know why
I am using this command

pcr.seqs(fasta=.\Ref\silva.bacteria.fasta, start=11894, end=25319, keepdots=F, processor=7)

As for the why, from the SOP:

"Now we need to align our sequences to the reference alignment. Again we can make our lives a bit easier by making a database customized to our region of interest using the pcr.seqs command. To run this command you need to have the reference database (silva.bacteria.fasta) and know where in that alignment your sequences start and end. To remove the leading and trailing dots we will set keepdots to false. You could also run this command using your primers of interest.

Those coordinates include the primers. The coordinates without the primers are 13862 and 23444. If you sequenced a region other than the V4 region, you should consult this blog post to see how to customize the coordinates for your region."

You can also use PCR.seq with your primers to get the region that specially cover your sequencing region.

From mothur wiki: "

The pcr.seqs will trim inputted sequences based on a variety of user-defined options.

The oligos file allows you to provide primer information.

mothur > pcr.seqs(fasta=silva.bacteria.fasta, oligos=pcrTest.oligos)


And you use the output file to align.

Also another tip: put all files, references, etc, in the same operating folder and run Mothur from that same folder.

Also, if you want help, how I am suppose to know how to help you if you are telling me that you have an error message but do not post the error? It is like telling you, please help me there is something wrong but I forget to tell you that I spilled my coffee and you show up with a broom. Use the log file to post the command that went wrong and the error shown.

please see the error

It basically say that Mothur is not finding the file. It might be an error in the name of the file or that the file is not there.

Please, see my past post where I recommend to put all files in the same directory and run Mothur from this directory. This way, you do not need to specify in which directory the files are in or wher the created files will be written.

Best of success,

well… kindly let me know how to find files location

kindly help me regarding this issue


Unfortunately, I do not have the time toinvest in this level of support as I fell you need more then just support on Mothur, but also in general bioinformatic skils.

I am truly sorry.

The best way is to put all files in the same folder (sequencing files, oligo files, reference files ( Silva reference files ( for taxonomy assignment, well all files) in the same folder as the mothur executable (if on Windows) or run Mothur from this directory (if you are on not Windows) and from there go on with your data analysis.

Sir… Well, I have solved that problem. Thank you so much for your guidance.
KIndly tell me on which parameter coordinates are selected in given commands

pcr.seqs(fasta= C:\Users\Rabia\Desktop\mothur\silva.bacteria\silva.bacteria.fasta, start=13862, end=23444, keepdots=F)

Best regards



What is your summary just after align.seq?