Issues with mothur opening a file

Hi, I’m kind of new to Mothur and am having some issues with the software not wanting to open my file.

Currently I have a folder named DUS_1 which is saved on my desktop. In this folder, I have Mothur as well as my fasta (file name = dusel) file with all my sequence data. The fasta file opens in Mega 4.0.2.

An example of a command I tried goes as follows

mothur > unique.seqs(fasta=DUS_1\dusel)

I had a good tutorial by a researcher who has used this quite a bit and this is how I thought I entered the command with how I have everything set up, but I keep getting a message that says "DUS_1\dusel could not be opened.

Does anyone have any insight to his???

Thanks a bunch folks

Jason Koch

Okay, so I’ve narrowed my problem down to the file type I am trying to input. The file type is a “FAS file” and it needs to be a “FASTA file.” I was under the assumption that FAS was the same as FASTA but apparently not.

Does anyone know how to convert to FASTA instead of FAS??

When I export the sequences form MEGA or Sequencher, I exported as a FASTA file but it gives me this FAS…



FAS and FASTA are the same thing. But… that doesn’t mean that is what you’ve got :slight_smile: A fasta file is a pretty simple format. For each sequence there is a header line that starts with a “>” character followed by the name of the sequence. The following lines are the sequences.

However, the error you’re getting makes me think that you may have the path wrong to the file. When you’re in mothur type system(“dir”). Do you see the DUS_1 folder? If so, then type: system(“dir DUS_1”). Do you see a file called dusel? I wonder if the file isn’t really dusel.fasta, dusel.fas, dusel.fa, or even dusel.txt - sometimes windows will hide the file extension.


Thanks Pat!

I actually resolved this by just saving the original sequencher export as dusel.fasta and i had no problem.

My new problem is that when I go to align my sequences to a reference database (silva) mothur will stop responding and I have to close it. I’m wondering if my computer (it’s a notebook) doesn’t have enough RAM to carry this out???

Let me know what you think.

It could be - you probably need at least 2 GB of RAM to use the silva alignment

I had the same problem, the alignment seemed to stop, I have to close it. I just did the alignment in Mega and skipped it in Mothur.


I think it would be in your best interest to figure out how to use something other than mega. It scales poorly and gives an inferior alignment for 16S sequences. When I review papers and people use clustal/muscle/mega I ding them on it.


Thanks a lot for the great guidance! Sorry for all the questions. Do you have any suggestions other than mega???

Thanks again!

Jason Koch

Jason - Datasets are really getting to the point where using a laptop is sadly, not often enough. Tell you PI that they dropped $10k on a dataset and they should splurge a few $$ on a computer. :slight_smile: