Hi I am Alan and I am new here, so sorry for asking stupid questions.
I have used Mothur to analyse a 454 sequenceing metagenome.
Everything went smoothly until to the final step when I looked at the an.unique_list.0.03.cons file, all the bacteria are classified well, except the problem is all the cyanobacteria are listed as unclassified below phylum level.
I used silva bacteria as alignment reference, and I used RDP as the classifying template.
Below is the pipeline of my analysis if it is required.
mothur > sffinfo(sff=pustular.sff, fasta=T)
mothur > make.group(fasta=pustular.fasta, groups=pustular)
mothur > screen.seqs(fasta=pustular.fasta, group=pustular.groups, maxambig=0, optimize=start-end, criteria=90, minlength=200, processors=4)
mothur > unique.seqs(fasta=pustular.good.fasta)
mothur > count.seqs(name=pustular.good.names, group=pustular.good.groups)
mothur > align.seqs(fasta=pustular.good.unique.fasta, reference=silva.bacteria.fasta)
mothur > screen.seqs(fasta=pustular.good.unique.align, count=pustular.good.count_table, summary=pustular.good.unique.summary, start=1044, end=8362)
mothur > filter.seqs(fasta=pustular.good.unique.good.align, vertical=T, trump=.)
mothur > unique.seqs(fasta=pustular.good.unique.good.filter.fasta, count=pustular.good.good.count_table)
mothur > pre.cluster(fasta=pustular.good.unique.good.filter.unique.fasta, count=pustular.good.unique.good.filter.count_table, diffs=3)
mothur > chimera.uchime(fasta=pustular.good.unique.good.filter.unique.precluster.fasta, count=pustular.good.unique.good.filter.unique.precluster.count_table, dereplicate=t)
mothur > remove.seqs(fasta=pustular.good.unique.good.filter.unique.precluster.fasta, accnos=pustular.good.unique.good.filter.unique.precluster.uchime.accnos)
mothur > classify.seqs(fasta=pustular.good.unique.good.filter.unique.precluster.pick.fasta, count=pustular.good.unique.good.filter.unique.precluster.uchime.pick.count_table, reference=trainset9_032012.rdp.fasta, taxonomy=trainset9_032012.rdp.tax)
mothur > dist.seqs(fasta=pustular.good.unique.good.filter.unique.precluster.pick.fasta, cutoff=0.30)
mothur > cluster(column=pustular.good.unique.good.filter.unique.precluster.pick.dist, count=pustular.good.unique.good.filter.unique.precluster.uchime.pick.count_table, cutoff=0.07)
mothur > make.shared(list=pustular.good.unique.good.filter.unique.precluster.pick.an.unique_list.list, count=pustular.good.unique.good.filter.unique.precluster.uchime.pick.count_table, label=0.03)
mothur > classify.otu(list=pustular.good.unique.good.filter.unique.precluster.pick.an.unique_list.list, count=pustular.good.unique.good.filter.unique.precluster.uchime.pick.count_table, taxonomy=pustular.good.unique.good.filter.unique.precluster.pick.rdp.wang.taxonomy, label=0.03)
Thanks in advance for helping!