Making a biom file for picrust

I know this question has been asked before, but i keep running into issues when trying to make a biom file for picrust.

I run classify.seqs, remove.lineage, and classify.otu. I run into problems with classify.otu where it says that my list file and group file don’t match.

The groups file i started using is SnailGut.contigs.good.groups and then i would use the new ones that were output.

The list I used at first was: SnailGut.trim.contigs.good.unique.good.filter.unique.precluster.pick.pick.opti_mcc.list
The first list file didn’t work and so i ran cluster.split with my current fasta, count, and green genes taxonomy file and got: SnailGut.trim.contigs.good.unique.good.filter.unique.precluster.pick.opti_mcc.list
Afterwards, I would use the pick.list file that was output.

mothur > classify.seqs(fasta=SnailGut.trim.contigs.good.unique.good.filter.unique.precluster.pick.fasta, template=/home/crharris/PICRUSt_Data/gg_13_5_99.fasta, taxonomy=/home/crharris/PICRUSt_Data/, name=SnailGut.trim.contigs.good.names, cutoff=97, group=SnailGut.contigs.good.groups)

mothur > cluster.split(fasta=SnailGut.trim.contigs.good.unique.good.filter.unique.precluster.pick.fasta, count=SnailGut.trim.contigs.good.unique.good.filter.unique.precluster.denovo.vsearch.pick.count_table,, splitmethod=classify, taxlevel=5, cutoff=0.03)

mothur > remove.lineage(, taxon=Chloroplast-Mitochondria-unknown-Eukaryota, name=SnailGut.trim.contigs.good.names, group=SnailGut.contigs.good.groups, list=SnailGut.trim.contigs.good.unique.good.filter.unique.precluster.pick.opti_mcc.list, label=0.03)

mothur > classify.otu(list=SnailGut.trim.contigs.good.unique.good.filter.unique.precluster.pick.opti_mcc.0.03.pick.list, name=SnailGut.trim.contigs.good.pick.names, group=SnailGut.contigs.good.pick.groups,, label=0.03)

Not sure what I’m doing wrong.

The mismatch is happening because you are using both a count file and name and group files in your analysis. The name and group file represent the same data as the count file, but mothur is designed to be used with one or the other, not both. When you create a list file with a names file all unique AND redundant sequences are included in the list file. When you create a list file using a count file, ONLY the unique names are included.

Instead try using these commands:

mothur > classify.seqs(fasta=SnailGut.trim.contigs.good.unique.good.filter.unique.precluster.pick.fasta, template=/home/crharris/PICRUSt_Data/gg_13_5_99.fasta, taxonomy=/home/crharris/PICRUSt_Data/, count=SnailGut.trim.contigs.good.unique.good.filter.unique.precluster.denovo.vsearch.pick.count_table) - classify sequences using the count file

mothur > cluster.split(fasta=current, count=current, taxonomy=current, splitmethod=classify, taxlevel=5, cutoff=0.03)

mothur > remove.lineage(taxonomy=current taxon=Chloroplast-Mitochondria-unknown-Eukaryota, count=current, list=current, label=0.03)

mothur > classify.otu(list=current, count=current, taxonomy=current, label=0.03)

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