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Downstream analysis with mothur

The starting point for downstream OTU analysis is an OTU table giving the number of reads per sample per OTU. This is all you need to perform many standard analyses including rarefaction, alpha diversity and beta diversity.

The mothur term for an OTU table is a "shared file". To get an OTU table in mothur shared file format, use the -mothur_shared_out option of usearch_global. See generating an OTU table for details.

You may also need a tree for the OTUs, e.g. for Unifrac analsysis as implemented in the unifrac.weighted and unifrac.unweighted commands in mothur. To make a tree, you can use the cluster_agg command in usearch. (Note there is a bug in this command in v9.0, you can work around it by using version 8 or version 9.1). For example:

usearch -cluster_agg otus.fa -treeout tree.phy

This generates a tree in Newick format which is compatible with mothur and many other programs. Some programs are very picky about labels in Newick files which may cause problems. To get the best compatibility with other software, you should update the sequence labels as follows. Delete any usearch-style annotations (things like size=123;). Make sure you use only letters and numbers, don't use anything else (even periods or underscores). Don't use labels longer than 10 characters. Labels like Otu123 should be compatible with most (all?) programs.

In the following examples I willl use the command-line mode of mothur. I'll assume the shared file is named shared.txt and is in the current directory when you start mothur. After running a mothur command as shown below, you should see the output files and a mothur log file in the current directory.

Rarefaction
You can use the rarefaction.single command.

mothur "#rarefaction.single(shared=shared.txt)"

Alpha diversity
You can use the summary.single command.

mothur "#summary.single(shared=shared.txt)"

Beta diversity
You can use the summary.shared command.

mothur "#summary.shared(shared=shared.txt)"