I am trying to use ANOSIM to compare two soil samples’ bacterial profiles. I currently follow the MiSeq protocol and I was hoping someone could give me pointers on when to run this command. I build a distance matrix (can be square or not), but I don’t know how to make a design file. I have tried making it using the file names I originally gave my sequences, but I always get an error message that says the files were not found.

My questions are:

  1. How do I make a proper design file?
  2. Do I need more than one file in each “group” of the design file.


A design file is basically just a ‘group of groups’ file. It looks the same as the groups file, but instead of clustering sequences into groups it clusters groups into bigger groups (or as I think of it, samples into treatment groups).

It’s just a tab-separated text file with your group names followed by treatment. E.g.

Group1 Treatment1
Group2 Treatment1
Group3 Treatment2
Group4 Treatment2

For your second question, if you're asking whether you need to have something like the above, rather than, say;

Group1 Treatment1
Group2 Treatment2
Group3 Treatment3
Group4 Treatment4

Then I would imagine the answer is yes, because ANOSIM is comparing your within- to between- group (treatment) variation so would require some replication to get a measure for the intra-treatment variation.