Design File error

mothur bugs
1

I’m running AMOVA on v.1.29.2 on a Mac version 10.5. I’m consistently getting 2 types of errors, whether or not I specify which sets to compare:
1.) Your designfile contains more than 1 sequence named 1, sequence names must be unique.
2.) [ERROR]: A6-7C20C30 is not in your design file, please correct. (This error then repeats for all of the remaining groups in my file).

The specific command I’m using is amova(phylip=final.an.0.03.subsample.thetayc.0.03.lt.dist, design=citratevspvp.design, sets=1-2)
I’ve tried making multiple changes to the formatting and content of my design file and still get these same errors. Could this be happening because I have dashes in my group names?

Thanks!

2

Could you try running set.dir(debug=t) before the amova command? It should output what mothur is reading when it reads your design file?

3

Yep - here’s what I got (I didn’t paste the whole thing as these errors basically repeat for each sample).

mothur > set.dir(debug=t)
Setting [DEBUG] flag.

mothur > amova(phylip=final.an.0.03.subsample.thetayc.0.03.lt.dist, design=citratevspvp.design, sets=1-2)
[DEBUG]: name = ‘{\rtf1\ansi\ansicpg1252\cocoartf949\cocoasubrtf540’, group = ‘{\fonttbl\f0\fnil\fcharset0’
[DEBUG]: name = ‘Monaco;}’, group = ‘{\colortbl;\red255\green255\blue255;}’
[DEBUG]: name = ‘\margl1440\margr1440\vieww9000\viewh8400\viewkind0’, group = ‘\pard\tx720\tx2160\tx2880\tx3600\tx4320\tx5040\tx5760\tx6480\tx7200\tx7920\tx8640\ql\qnatural\pardirnatural’
[DEBUG]: name = ‘\f0\fs20’, group = ‘\cf0’
[DEBUG]: name = ‘A1-1C20C25’, group = ‘1’
[DEBUG]: name = ‘A10-7C20H46’, group = ‘1’
[DEBUG]: name = ‘A11-7C20H47’, group = ‘1’
[DEBUG]: name = ‘A2-1C20C26’, group = ‘1’
[DEBUG]: name = ‘A3-1C20C27’, group = ‘1’
[DEBUG]: name = ‘A4-7C20C28’, group = ‘1’
[DEBUG]: name = ‘A5-7C20C29’, group = ‘1’
[DEBUG]: name = ‘’, group = ‘A6-7C20C30’
[DEBUG]: name = ‘1’, group = ‘A7-1C20H43’
[DEBUG]: name = ‘1’, group = ‘A8-1C20H44’
Your designfile contains more than 1 sequence named 1, sequence names must be unique. Please correct.
[DEBUG]: name = ‘1’, group = ‘A9-1C20H45’
Your designfile contains more than 1 sequence named 1, sequence names must be unique. Please correct.
[DEBUG]: name = ‘1’, group = ‘B1-7C20H48’
Your designfile contains more than 1 sequence named 1, sequence names must be unique. Please correct.
[DEBUG]: name = ‘1’, group = ‘B11-1C110H67’
Your designfile contains more than 1 sequence named 1, sequence names must be unique. Please correct.
[DEBUG]: name = ‘1’, group = ‘B12-1C110H68’
Your designfile contains more than 1 sequence named 1, sequence names must be unique. Please correct.
[DEBUG]: name = ‘1’, group = ‘B2-1C110C49’
Your designfile contains more than 1 sequence named 1, sequence names must be unique. Please correct.
[DEBUG]: name = ‘2’, group = ‘’
[DEBUG]: name = ‘D9-1P110C145’, group = ‘2’
[DEBUG]: name = ‘E1-1P20H139’, group = ‘2’
[DEBUG]: name = ‘E10-7P110H167’, group = ‘2’
[DEBUG]: name = ‘E11-7P110H168’, group = ‘2’
[DEBUG]: name = ‘E2-1P20H140’, group = ‘2’
[DEBUG]: name = ‘E3-7P110C149’, group = ‘2’
[DEBUG]: name = ‘E4-7P110C150’, group = ‘2’
[DEBUG]: name = ‘E6-1P110H163’, group = ‘2’
[DEBUG]: name = ‘E7-1P110H164’, group = ‘2’
[DEBUG]: name = ‘E8-1P110H165’, group = ‘2’
[DEBUG]: name = ‘E9-7P110H166’, group = ‘2’
[DEBUG]: name = ‘’, group = ‘}’
Your designfile contains more than 1 sequence named , sequence names must be unique. Please correct.
2 is not a valid group, and will be disregarded.
[ERROR]: A6-7C20C30 is not in your design file, please correct.

4

Ah - I see the problem now! There were extra spaces after my group names that I couldn’t see when actually looking at my design file, so the system was using my group values as name values for some of the samples and finding duplicates. I fixed that in my design file and now amova is working fine. Thanks a lot - that debug command is super helpful!

5

Glad to help, :slight_smile: