Interactive mode

Default mode

When you start mothur, you will see a screen that looks like this:

mothur v1.0
Last updated: 1/29/2009

Patrick D. Schloss

Department of Microbiology
The University of Massachusetts [``](

Distributed under the GNU General Public License

Type 'help()' for information on the commands that are available

Type 'quit()' to exit program

mothur >

All of your commands will be entered from the mothur prompt. You can get a list of commands by typing help() at the prompt and you can get more specific help for each command by typing command_name(help) at the prompt. For example:

mothur > align.seqs(help)

Would give you the help file information for the align.seqs command.

If you want to quit mothur you simply enter:

mothur > quit()


mothur > quit

With the exception of quit, all commands require you to provide an open and close parentheses. If you supply any options there cannot be a space between the option, the ‘=’, and the option setting. To separate options, use a comma. For example:

mothur > cluster(method=furthest, cutoff=0.10)

Environment Variables

Environment variables can be used to generalize variables for reuse. Let’s add environment variables to our analysis.


The format of environmental variables is [tag]=[value]. For example, let’s create a environment variable for the processors option.


mothur will automatically pull in the systems environment variable. For example you can set variables in bash and then run mothur:

. mothurMac-3% export REFERENCE_LOCATION=/Users/sarahwestcott/Desktop/release . mothurMac-3% export ALIGNREF=silva.v4.fasta . mothurMac-3% export TAXONREF_FASTA=trainset9_032012.pds.fasta . mothurMac-3% export . mothurMac-3% export CONTAMINENTS=Chloroplast-Mitochondria-unknown-Archaea-Eukaryota . mothurMac-3% export LOGNAME=MiSEQ_SOP_mouse_03032020 . mothurMac-3% export DATA=/Users/sarahwestcott/Desktop/MiSeq_SOP . mothurMac-3% export TYPE=gz . mothurMac-3% export PROC=12 . mothurMac-3% ./mothur 

Type 'quit()' to exit program

[note]: Setting random seed to 19760620.

mothur >  set.logfile(name=$LOGNAME)

mothur > set.dir(input=$DATA)   . mothur's directories:


The ‘$’ symbol indicates to mothur that the value is an environment variable to be replaced with the actual value at run time. For example:

make.contigs(file=current, processors=$PROC)

is equivalent to

make.contigs(file=current, processors=12)

You can create an environment variable for anything you wish as well as combining variables. For example:

mothur > REFERENCE_LOCATION=/Users/sarahwestcott/Desktop/release
Setting environment variable REFERENCE_LOCATION to /Users/sarahwestcott/Desktop/release

mothur > ALIGNREF=silva.v4.fasta
Setting environment variable ALIGNREF to silva.v4.fasta

mothur > align.seqs(fasta=my.fasta, reference=$REFERENCE_LOCATION/$ALIGNREF)

Alternatively, If you know what commands you will be running, you can use either the command line mode or batch mode.