Currently, I run my MiSeq PE through mothur following MiSeq SOP. Everything seems fine before making OTUs. When I make OTUs (make.shared) and taxonomic classification (classify.otu), mothur told me ‘Your file does not include the label 0.03. I will use unique.’ By the way, I used mothur.v.1.33.2
To make OTUs, I followed MiSeq SOP, as following:
mothur>dist.seqs(fasta=stability.trim.contigs.good.unique.good.filter.unique.precluster.pick.pick.fasta, cutoff=0.20)
mothur>cluster(column=stability.trim.contigs.good.unique.good.filter.unique.precluster.pick.pick.dist,count=stability.trim.contigs.good.unique.good.filter.unique.precluster.uchime.pick.pick.count_table)
mothur>make.shared(list=stability.trim.contigs.good.unique.good.filter.unique.precluster.pick.pick.an.unique_list.list,count=stability.trim.contigs.good.unique.good.filter.unique.precluster.uchime.pick.pick.count_table, label=0.03)
mothur>classify.otu(list=stability.trim.contigs.good.unique.good.filter.unique.precluster.pick.pick.an.unique_list.list,count=stability.trim.contigs.good.unique.good.filter.unique.precluster.uchime.pick.pick.count_table,taxonomy=stability.trim.contigs.good.unique.good.filter.unique.precluster.pick.pds.wang.pick.taxonomy, label=0.03)
When I run MiSeq SOP examples, those two commands recognize ‘label=0.03’, and but not for my own data. I couldn’t figure out what cause the problem.
Any suggestions will be helpful. Thank you very much in advance.
Just add more information. Before I started making OTUs, I run
mothur>dist.seqs(fasta=stability.trim.contigs.good.unique.good.filter.unique.precluster.pick.pick.fasta, cutoff=0.20)
mothur>cluster(column=stability.trim.contigs.good.unique.good.filter.unique.precluster.pick.pick.dist,count=stability.trim.contigs.good.unique.good.filter.unique.precluster.uchime.pick.pick.count_table)
After this step, it generated file called ‘stability.trim.contigs.good.unique.good.filter.unique.precluster.pick.pick.an.unique_list.list’, which has several rows indicating label from ‘unique’, ‘0.01’, …, ‘0.08’. For somehow, I only have two rows in this file, one is titles, and another row is ‘unique’. It doesn’t have other label, which caused the next step for make.shared() couldn’t fine ‘label=0.03’
At last, I tried alternative way to cluster for MiSeq, it works fine:
mothur<cluster.split(fasta=stability.trim.contigs.good.unique.good.filter.unique.precluster.pick.pick.fasta,count=stability.trim.contigs.good.unique.good.filter.unique.precluster.uchime.pick.pick.count_table,taxonomy=stability.trim.contigs.good.unique.good.filter.unique.precluster.pick.pds.wang.pick.taxonomy, splitmethod=classify, taxlevel=4, cutoff=0.15, processors=16)
I couldn’t figure out why cluster() didn’t give me more than unique clusters. cluster.split gives ‘unique, 0.01,0.02,0.03,0.04,0.05’ label. But why there is no ‘0.08’ label?
Thank you very much for your help. I read the FAQ, which helped.
The output from dist.seqs and cluster:
mothur > dist.seqs(fasta=stability.trim.contigs.good.unique.good.filter.unique.precluster.pick.pick.fasta, cutoff=0.20)
My distance file from dist.seqs is about 100GB. I guess mothur wasn’t able to read the whole distance file, but I didn’t get any warning.
When I use classify.split(), I set cutoff=0.15, which helps for large data. At the end of running classify.split, it said ‘change cutoff to 0.05’.
stability.trim.contigs.good.unique.good.filter.unique.precluster.pick.pick.fasta.0.dist
Cutoff was 0.155 changed cutoff to 0.05
Cutoff was 0.155 changed cutoff to 0.05
Cutoff was 0.155 changed cutoff to 0.05
It took 22514 seconds to cluster
Merging the clustered files…
It took 10 seconds to merge.