make.shared not executed

Commands in mothur
1

Dear all,

I followed the 454 SOP. However, when I run make.shared(list=merged.unique.good.filter.unique.precluster.pick.pick.an.list, group=merged.good.pick.pick.groups, label=0.03) the command doesnt get executed. The .shared file in my directiory is empty and theres no .rabund file.

I already ran this command before without troubles. The only thing I changed this time, is the reference database. I used silva.nr_v119.ng.fasta before, and this time I`d like to use silva.nr_v119.fasta.

Because silva.nr_v119.fasta is bigger than silva.nr_v119.ng.fasta I wonder, if the command does not work because of lack of memory? Or does this even have an association? Or anyone has any other ideas?

Any kind of help appreciated! Thank you.

2

Can you post the exact commands you are running and the error message you are getting?

3

this is my script:

mothur > make.group(fasta=merged.fasta, groups=A)
mothur > unique.seqs(fasta=merged.fasta)
mothur > align.seqs(fasta=merged.unique.fasta, reference=silva.nr_v119.align, processors=2)
mothur > screen.seqs(fasta=merged.unique.align, name=merged.names, group=merged.groups, end=27654, optimize=start, criteria=95, processors=2)
mothur > filter.seqs(fasta=merged.unique.good.align, vertical=T, trump=., processors=2)
mothur > unique.seqs(fasta=merged.unique.good.filter.fasta, name=merged.good.names)
mothur > pre.cluster(fasta=merged.unique.good.filter.unique.fasta, name=merged.unique.good.filter.names, group=merged.good.groups, diffs=2)
mothur > chimera.uchime(fasta=merged.unique.good.filter.unique.precluster.fasta, name=merged.unique.good.filter.unique.precluster.names, group=merged.good.groups, processors=2)
mothur > remove.seqs(accnos=merged.unique.good.filter.unique.precluster.uchime.accnos, fasta=merged.unique.good.filter.unique.precluster.fasta, name=merged.unique.good.filter.unique.precluster.names,group=merged.good.groups, dups=T)
mothur > classify.seqs(fasta=merged.unique.good.filter.unique.precluster.pick.fasta, name=merged.unique.good.filter.unique.precluster.pick.names, group=merged.good.pick.groups, template=silva.nr_v119.align, taxonomy=silva.nr_v119.tax, processors=2)
mothur > remove.lineage(fasta=merged.unique.good.filter.unique.precluster.pick.fasta, name=merged.unique.good.filter.unique.precluster.pick.names, group=merged.good.pick.groups, taxonomy=merged.unique.good.filter.unique.precluster.pick.nr_v119.wang.taxonomy, taxon=Mitochondria-Chloroplast-Archaea-Eukaryota-unknown)
mothur > dist.seqs(fasta=merged.unique.good.filter.unique.precluster.pick.pick.fasta, cutoff=0.15, processors=2)
mothur > cluster(column=merged.unique.good.filter.unique.precluster.pick.pick.dist, name=merged.unique.good.filter.unique.precluster.pick.pick.names)
mothur > make.shared(list=merged.unique.good.filter.unique.precluster.pick.pick.an.list, group=merged.good.pick.pick.groups, label=0.03)

I don`t get an error message printed to the console. However, I just had a look at the Logfile, where it says that sequences are in my groupfile, but not in the listfile. Am I using the wrong .groups file? Seems a little strange to me, since -as said- the exact same code worked before.

4

Do you only have one group - A ? If so, you don’t need a shared file since it would be the same as the list file.

5

Ok, I got it that my .groups file is wrong.
What I actually would need is a .groups file that contains all my sample names.
Is there a way to do it without the trim.seqs step, where I originally in a previous run included the .oligos files indicating the sample names?

Thank you very much!

6

Is there a way to do it without the trim.seqs step, where I originally in a previous run included the .oligos files indicating the sample names?

I’m not sure what this means… You can make a groups file like you did with make.group, but by chaining the fasta and group names together…

 make.group(fasta=sample1.fasta-sample2.fasta-sample3.fasta, groups=A-B-C)
7

Alright!
Thanks a lot for your help!