mothur

Classify.seq could not be classified

Hi I am trying to run a classify.seq command using the silva database as a reference however I am getting a warning saying [WARNING]: M05510_34_000000000-JD7WL_1_2119_20097_23995 could not be classified. You can use the remove.lineage command with taxon=unknown; to remove such sequences, this is for all my bacteria sequences and when I quit the command it says “all sequences have been removed”. I was using this version of mothur before mothur v.1.41.3 and then changed to this one (mothur v.1.44.3) but I’m still getting the same issue. Please kindly assist.

Hi,

Can you post one of the sequences so I can take a look at it?

Thanks,
Pat

Hello Pat.

I was advised to let it run until it finishes, which took a while but I did manage to get a taxonomy file at the end. Apparently, it’s normal for it to do that.

kind regards

Nicky

Hie Pat

I wanted to ask if you could help, when I run my dist.seqs command it says that it is blank and this might be that there are are no distances below my cutoff. I used 0.03.

Kind regards

Nicky

What are you sequencing? Can you post your commands?

I’m sequencing bacteria, here are my commands

#make.contigs(file=bacteria.files)
#summary.seqs(fasta=bacteria.trim.contigs.fasta, processors=24)
#trim.seqs(fasta=bacteria.trim.contigs.fasta, maxambig=0, minlength=350, maxlength=550)

#summary.seqs(fasta=bacteria.trim.contigs.trim.fasta)
#unique.seqs(fasta=bacteria.trim.contigs.trim.fasta)
#summary.seqs(fasta=bacteria.trim.contigs.trim.unique.fasta, name=bacteria.trim.contigs.trim.names)
#count.seqs(name=bacteria.trim.contigs.trim.names, group=bacteria.contigs.groups)
#summary.seqs(fasta=bacteria.trim.contigs.trim.unique.fasta, name=bacteria.trim.contigs.trim.names)
#chimera.vsearch(fasta=bacteria.trim.contigs.trim.unique.fasta, count=bacteria.trim.contigs.trim.count_table, dereplicate=t)
#remove.seqs(fasta=bacteria.trim.contigs.trim.unique.fasta,accnos=bacteria.trim.contigs.trim.unique.denovo.vsearch.accnos)
#summary.seqs(fasta=bacteria.trim.contigs.trim.unique.pick.fasta, count=bacteria.trim.contigs.trim.denovo.vsearch.pick.count_table, processors=24)
#classify.seqs(fasta=bacteria.trim.contigs.trim.unique.pick.fasta, count=bacteria.trim.contigs.trim.denovo.vsearch.pick.count_table, reference=silva.nr_v138_1.align, taxonomy=silva.nr_v138_1.tax, cutoff=80)
#remove.lineage(fasta=bacteria.trim.contigs.trim.unique.pick.fasta, count=bacteria.trim.contigs.trim.denovo.vsearch.pick.count_table, taxonomy=bacteria.trim.contigs.trim.unique.pick.nr_v138_1.wang.taxonomy, taxon=Chloroplast-Mitochondria-unknown-Archaea-Eukaryota)
#summary.seqs(fasta=bacteria.trim.contigs.trim.unique.pick.pick.fasta, count=bacteria.trim.contigs.trim.denovo.vsearch.pick.pick.count_table, processors=24)
#summary.tax(taxonomy=bacteria.trim.contigs.trim.unique.pick.nr_v138_1.wang.pick.taxonomy, count=bacteria.trim.contigs.trim.denovo.vsearch.pick.pick.count_table)
#align.seqs(fasta=bacteria.trim.contigs.trim.unique.pick.pick.fasta, reference=silva.nr_v138_1.align, flip=T)
#summary.seqs(fasta=bacteria.trim.contigs.trim.unique.pick.pick.fasta, count=bacteria.trim.contigs.trim.denovo.vsearch.pick.pick.count_table)
#summary.seqs(fasta=bacteria.trim.contigs.trim.unique.pick.pick.align,count=bacteria.trim.contigs.trim.denovo.vsearch.pick.pick.count_table, processors=24)

#screen.seqs(fasta=bacteria.trim.contigs.trim.unique.pick.pick.align, count=bacteria.trim.contigs.trim.denovo.vsearch.pick.pick.count_table, start=11895 end=28464)
#summary.seqs(fasta=bacteria.trim.contigs.trim.unique.pick.pick.good.align, count=bacteria.trim.contigs.trim.denovo.vsearch.pick.pick.good.count_table)
#filter.seqs(fasta=bacteria.trim.contigs.trim.unique.pick.pick.good.align, vertical=T, trump=.)
#count.groups(count=bacteria.trim.contigs.trim.denovo.vsearch.pick.pick.good.count_table)
#sub.sample(fasta= bacteria.trim.contigs.trim.unique.pick.pick.good.filter.fasta, count=bacteria.trim.contigs.trim.denovo.vsearch.pick.pick.good.count_table, size=24804, persample=t)
#count.groups(count=bacteria.trim.contigs.trim.denovo.vsearch.pick.pick.good.subsample.count_table)
#unique.seqs(fasta=bacteria.trim.contigs.trim.unique.pick.pick.good.filter.subsample.fasta, count=bacteria.trim.contigs.trim.denovo.vsearch.pick.pick.good.subsample.count_table)
#classify.seqs(fasta=bacteria.trim.contigs.trim.unique.pick.pick.good.filter.subsample.unique.fasta, #count=bacteria.trim.contigs.trim.unique.pick.pick.good.filter.subsample.count_table, reference=silva.nr_v138_1.align, taxonomy=silva.nr_v138_1.tax, cutoff=80)
#dist.seqs(fasta=bacteria.trim.contigs.trim.unique.pick.pick.good.filter.subsample.unique.fasta, cutoff=0.03)

I assume 16S? What region? Can you include the output from running summary.seqs at each of the steps you have it listed above?

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