Interactive mode

Default mode

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

Mac version

Using ReadLine,Boost,HDF5,GSL
mothur v.1.45.0
Last updated: 3/22/21
Patrick D. Schloss

Department of Microbiology & Immunology

University of Michigan

When using, please cite:
Schloss, P.D., et al., Introducing mothur: Open-source, platform-independent, community-supported software for describing and comparing microbial communities. Appl Environ Microbiol, 2009. 75(23):7537-41.

Distributed under the GNU General Public License

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

For questions and analysis support, please visit our forum at

Type 'quit()' to exit program

[NOTE]: Setting random seed to 19760620.

Interactive Mode

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=opti, cutoff=0.03)

Running a batch file

You can run a batch file from the mothur prompt as follows:

mothur > stability.batch

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.