Phylotype analyses: make.shared produces no output

Commands in mothur
1

Hey,
I’m almost done with my protist community analysis. I wanted to look at phylotypes but the following things happen.
1.) After the phylotype command, my list file contains 19 OTUs only.
and 2.) when I run make.shared, it seems to work (although there is a discrepancy between the number of sequences in the count file and the list) but the output files are just not generated. Has this happened to anyone else? Any idea on what I could try to make it work?

phylotype(taxonomy=deci_0.1_stability.trim.contigs.good.unique.good.filter.precluster.pick.silva.wang.pick.pick.taxonomy, label=4)

4

Output File Names:
deci_0.1_stability.trim.contigs.good.unique.good.filter.precluster.pick.silva.wang.pick.pick.tx.sabund
deci_0.1_stability.trim.contigs.good.unique.good.filter.precluster.pick.silva.wang.pick.pick.tx.rabund
deci_0.1_stability.trim.contigs.good.unique.good.filter.precluster.pick.silva.wang.pick.pick.tx.list

make.shared(list=deci_0.1_stability.trim.contigs.good.unique.good.filter.precluster.pick.silva.wang.pick.pick.tx.list, count=deci_0.1_stability.trim.contigs.good.unique.good.filter.precluster.uchime.pick.pick.pick.count_table, label=4)

Output File Names:
deci_0.1_stability.trim.contigs.good.unique.good.filter.precluster.pick.silva.wang.pick.pick.tx.shared
deci_0.1_stability.trim.contigs.good.unique.good.filter.precluster.pick.silva.wang.pick.pick.tx.110_1.rabund
deci_0.1_stability.trim.contigs.good.unique.good.filter.precluster.pick.silva.wang.pick.pick.tx.110_2.rabund
deci_0.1_stability.trim.contigs.good.unique.good.filter.precluster.pick.silva.wang.pick.pick.tx.110_3.rabund
deci_0.1_stability.trim.contigs.good.unique.good.filter.precluster.pick.silva.wang.pick.pick.tx.20_1.rabund
deci_0.1_stability.trim.contigs.good.unique.good.filter.precluster.pick.silva.wang.pick.pick.tx.20_2.rabund
deci_0.1_stability.trim.contigs.good.unique.good.filter.precluster.pick.silva.wang.pick.pick.tx.20_3.rabund
deci_0.1_stability.trim.contigs.good.unique.good.filter.precluster.pick.silva.wang.pick.pick.tx.65_1.rabund
deci_0.1_stability.trim.contigs.good.unique.good.filter.precluster.pick.silva.wang.pick.pick.tx.65_2.rabund
deci_0.1_stability.trim.contigs.good.unique.good.filter.precluster.pick.silva.wang.pick.pick.tx.65_3.rabund
deci_0.1_stability.trim.contigs.good.unique.good.filter.precluster.pick.silva.wang.pick.pick.tx.75_1.rabund
deci_0.1_stability.trim.contigs.good.unique.good.filter.precluster.pick.silva.wang.pick.pick.tx.75_3.rabund
deci_0.1_stability.trim.contigs.good.unique.good.filter.precluster.pick.silva.wang.pick.pick.tx.85_1.rabund
deci_0.1_stability.trim.contigs.good.unique.good.filter.precluster.pick.silva.wang.pick.pick.tx.85_2.rabund
deci_0.1_stability.trim.contigs.good.unique.good.filter.precluster.pick.silva.wang.pick.pick.tx.85_3.rabund

2

So the list file only contains the unique sequence names, their abundances are stored in the count file that is used in make.shared.

the output files are just not generated

can you tell me what you mean by this? are the files not there? are any error messages given?

Pat

3

Hey Pat,
thank you for helping!
I mean that although according to the prompt, the files are generated, they aren’t. In the folder, there is a new mothur-logfile but no rabund or shared files. And they aren’t in any other folder either.
Cheers,
Karin

4

I forgot:I don’t really get an error message, but there is an incongruence between the count file (containing more sequences) and the list file.

[ERROR]: MISEQ_90_000000000-A73ND_1_2114_9998_22336 is in your groupfile and not your listfile. Please correct.
[ERROR]: MISEQ_90_000000000-A73ND_1_2114_9998_24211 is in your groupfile and not your listfile. Please correct.
[ERROR]: MISEQ_90_000000000-A73ND_1_2114_9998_24741 is in your groupfile and not your listfile. Please correct.
[ERROR]: MISEQ_90_000000000-A73ND_1_2114_9998_3251 is in your groupfile and not your listfile. Please correct.
[ERROR]: MISEQ_90_000000000-A73ND_1_2114_9999_21466 is in your groupfile and not your listfile. Please correct.
[ERROR]: MISEQ_90_000000000-A73ND_1_2114_9999_22560 is in your groupfile and not your listfile. Please correct.
[ERROR]: MISEQ_90_000000000-A73ND_1_2114_9999_26983 is in your groupfile and not your listfile. Please correct.
[ERROR]: MISEQ_90_000000000-A73ND_1_2114_9999_3894 is in your groupfile and not your listfile. Please correct.
[ERROR]: MISEQ_90_000000000-A73ND_1_2114_9999_3944 is in your groupfile and not your listfile. Please correct.
[ERROR]: MISEQ_90_000000000-A73ND_1_2114_9999_4708 is in your groupfile and not your listfile. Please correct.
…

I started the analysis with 10% of my total sequences. These errors above did not affect the make.shared command when I did the OTU based runs.
Kind regards,
Karin

5

So those are error messages!

I’m not sure why make.shared would work with OTUs but not with phylotypes. Can you tell me how many lines are in the taxonomy file that goes into phylotype and how many lines are in the count_table file? If you’re doing this in mac/linux you can just do:

wc -l deci_0.1_stability.trim.contigs.good.unique.good.filter.precluster.pick.silva.wang.pick.pick.taxonomy
wc -l deci_0.1_stability.trim.contigs.good.unique.good.filter.precluster.uchime.pick.pick.pick.count_table

6

Hey Pat,
here’s the number of lines:
77271 deci_0.1_stability.trim.contigs.good.unique.good.filter.precluster.pick.silva.wang.pick.pick.taxonomy
3706564 deci_0.1_stability.trim.contigs.good.unique.good.filter.precluster.uchime.pick.pick.pick.count_table
Cheers,
Karin

7

Yeah there’s something wrong, you should be getting about 77272 lines in your count_table at this point. Can you post all of your commands?