ANOSIM and design files

Commands in mothur
1

Good afternoon!

I am trying to run an ANOSIM for the first time (v 1.29 on 64-bit Windows) and have run into unexpected difficulties with creating and using a design file (I’ve not made a design file before, either). I saw that I could create the file from my groups, and opened and looked at your Dunes example, so I created a tab-deliminated file for each group and treatment like this:

3245S_HIGH High
3016S_LOW Low
3246S_HIGH High
3231S_LOW Low
3039S_HIGH High
3091S_LOW Low
3438S_HIGH High
3446S_LOW Low
3245L_HIGH High
3016L_LOW Low
3246L_HIGH High
3231L_LOW Low
3039L_HIGH High
3091L_LOW Low
3438L_HIGH High
3446L_LOW Low

And ran the the command: mothur > anosim(phylip=Cows4.final.phylip.dist, design=HighvsLowGroups.design)

But running it gave me the errors:

[ERROR]: HOMLQBF01DOXMU is not in your design file, please correct.
[ERROR]: HOMLQBF01BZJJY is not in your design file, please correct.
etc.

for what appears to be each sequence in my file. As far as I can tell I do not have any extra spaces in my design file after the group names (I saw Design File error), and I am confused as to why it would be looking for sequences in that file, since it should just be groups and treatments? I ran the debug command (same previous post) and get:

[DEBUG]: name = ‘3245S_HIGH’, group = ‘High’
[DEBUG]: name = ‘3016S_LOW’, group = ‘Low’
[DEBUG]: name = ‘3246S_HIGH’, group = ‘High’
[DEBUG]: name = ‘3231S_LOW’, group = ‘Low’
[DEBUG]: name = ‘3039S_HIGH’, group = ‘High’
[DEBUG]: name = ‘3091S_LOW’, group = ‘Low’
[DEBUG]: name = ‘3438S_HIGH’, group = ‘High’
[DEBUG]: name = ‘3446S_LOW’, group = ‘Low’
[DEBUG]: name = ‘3245L_HIGH’, group = ‘High’
[DEBUG]: name = ‘3016L_LOW’, group = ‘Low’
[DEBUG]: name = ‘3246L_HIGH’, group = ‘High’
[DEBUG]: name = ‘3231L_LOW’, group = ‘Low’
[DEBUG]: name = ‘3039L_HIGH’, group = ‘High’
[DEBUG]: name = ‘3091L_LOW’, group = ‘Low’
[DEBUG]: name = ‘3438L_HIGH’, group = ‘High’
[DEBUG]: name = ‘3446L_LOW’, group = ‘Low’
[ERROR]: HOMLQBF01DOXMU is not in your design file, please correct.
[ERROR]: HOMLQBF01BZJJY is not in your design file, please correct.
[ERROR]: HOMLQBF01CFJY3 is not in your design file, please correct.
etc.

Please, I know I must be doing something simple and silly … but I really need help spotting it!

Thank you!

-Kelsea Jewell

2

Does your phylip file contain distances between sequences or groups?

3

I used dist.seqs on my post-classification fasta to generate my distance matrix (dist.seqs(fasta=Cows4.final.fasta, output=lt), run after removing chimeras, classifying, and removing lineages). I am assuming that method is between sequences, since there isn’t a group file specified. Am I misunderstanding this command? The output from dist.seqs (Cows4.final.phylip.dist) is my input for the ANOSIM command. I do have a group file (Cows4.final.groups) that I have used for my other commands, and was processed with those sequences.

4

Well, it took me a week and some but I managed to work through my design file issues. It turned out that an error must have occurred when writing a file, because after much experimentation I solved the problem by re-running my commands to rebuild all of the pertinent files. I still don’t know what was wrong … but at least now I have been successfully using design files!

5

Hi Pat and Sarah,

I am having the same issue except I have not been able to resolve it.

I am getting two errors. First I get a bunch of this:

[WARNING]: group ¢( 5í‚Ç*Q>Àēƪc[žii contains illegal characters in the name. Group names should not include :, -, or / characters. The ‘:’ character is a special character used in trees. Using ‘:’ will result in your tree being unreadable by tree reading software. The ‘-’ character is a special character used by mothur to parse group names. Using the ‘-’ character will prevent you from selecting groups. The ‘/’ character will created unreadable filenames when mothur includes the group in an output filename.

I am not using any of these characters.

Second I get this:
[ERROR]: group M03132_143_000000000-CBV6Y_1_1101_13438_1594 is not in your design file. Please correct.
[ERROR]: M03132_143_000000000-CBV6Y_1_1101_13438_1594 is not in your design file, please correct.
[ERROR]: group M03132_143_000000000-CBV6Y_1_1101_15985_1494 is not in your design file. Please correct.
[ERROR]: M03132_143_000000000-CBV6Y_1_1101_15985_1494 is not in your design file, please correct.
[ERROR]: group M03132_143_000000000-CBV6Y_1_1101_15331_3446 is not in your design file. Please correct.
[ERROR]: M03132_143_000000000-CBV6Y_1_1101_15331_3446 is not in your design file, please correct.
[ERROR]: group M03132_143_000000000-CBV6Y_1_1101_18190_12669 is not in your design file. Please correct.
[ERROR]: M03132_143_000000000-CBV6Y_1_1101_18190_12669 is not in your design file, please correct.
[ERROR]: group M03132_143_000000000-CBV6Y_1_1101_9144_8770 is not in your design file. Please correct.
[ERROR]: M03132_143_000000000-CBV6Y_1_1101_9144_8770 is not in your design file, please correct.
[ERROR]: group M03132_143_000000000-CBV6Y_1_1101_12169_5245 is not in your design file. Please correct.

I am totally stumped. Please help!

6

Would you mind posting this question in a new thread? This one is about 6 years old. If you could include the contents of your design file that would be helpful.

Pat