In the make.shared step, the label is set to 0.03, but the program always shows that it runs in unique. My goal is to cluster to OTU, but it can only be ASV. I checked the list file, only unique. I don’t know why, and which step is wrong?
Hi,
Can you provide the commands the the syntax you are uing before make.shared along with the syntax of make.shared?
Thanks,
Pat
make.file(inputdir=sampleY, type=fastq, prefix=stability)
make.contigs(file=stability.files, processors=8)
sub.sample(fasta=stability.trim.contigs.fasta,group=stability.contigs.groups,persample=T,size=10000)
summary.seqs(fasta=stability.trim.contigs.subsample.fasta)
screen.seqs(fasta=stability.trim.contigs.subsample.fasta,group=stability.contigs.subsample.groups, maxambig=0, maxlength=420)
unique.seqs(fasta=stability.trim.contigs.subsample.good.fasta)
align.seqs(fasta=stability.trim.contigs.subsample.good.unique.fasta,reference=silva.nr_v138.align)
summary.seqs(fasta=stability.trim.contigs.subsample.good.unique.align,count=stability.trim.contigs.subsample.good.count_table)
screen.seqs(fasta=stability.trim.contigs.subsample.good.unique.align,count=stability.trim.contigs.subsample.good.count_table,summary=stability.trim.contigs.subsample.good.unique.summary,start=4,end=14575,maxhomop=8)
filter.seqs(fasta=stability.trim.contigs.subsample.good.unique.good.align,vertical=T,trump=.)
unique.seqs(fasta=stability.trim.contigs.subsample.good.unique.good.filter.fasta,count=stability.trim.contigs.subsample.good.good.count_table)
pre.cluster(fasta=stability.trim.contigs.subsample.good.unique.good.filter.unique.fasta,count=stability.trim.contigs.subsample.good.unique.good.filter.count_table, diffs=2)
chimera.vsearch(fasta=stability.trim.contigs.subsample.good.unique.good.filter.unique.precluster.fasta,count=stability.trim.contigs.subsample.good.unique.good.filter.unique.precluster.count_table,dereplicate=t)
remove.seqs(fasta=stability.trim.contigs.subsample.good.unique.good.filter.unique.precluster.fasta,accnos=stability.trim.contigs.subsample.good.unique.good.filter.unique.precluster.denovo.vsearch.accnos)
classify.seqs(fasta=stability.trim.contigs.subsample.good.unique.good.filter.unique.precluster.pick.fasta,count=stability.trim.contigs.subsample.good.unique.good.filter.unique.precluster.denovo.vsearch.pick.count_table,reference=silva.nr_v138.align,taxonomy=silva.nr_v138.tax,cutoff=80)
remove.lineage(fasta=stability.trim.contigs.subsample.good.unique.good.filter.unique.precluster.pick.fasta,count=stability.trim.contigs.subsample.good.unique.good.filter.unique.precluster.denovo.vsearch.pick.count_table,taxonomy=stability.trim.contigs.subsample.good.unique.good.filter.unique.precluster.pick.nr_v138.wang.taxonomy, taxon=Chloroplast-Mitochondria-unknown-Eukaryota)
dist.seqs(fasta=stability.trim.contigs.subsample.good.unique.good.filter.unique.precluster.pick.pick.fasta,cutoff=0.1)
cluster(column=stability.trim.contigs.subsample.good.unique.good.filter.unique.precluster.pick.pick.dist,count=stability.trim.contigs.subsample.good.unique.good.filter.unique.precluster.denovo.vsearch.pick.pick.count_table,method=average)
make.shared(list=stability.trim.contigs.subsample.good.unique.good.filter.unique.precluster.pick.pick.an.list,count=stability.trim.contigs.subsample.good.unique.good.filter.unique.precluster.denovo.vsearch.pick.pick.count_table,label=0.03)
Thanks！
I think the problem is because you are using method=average in the cluster command. I’d strongly recommend removing the method=average argument and changing the cutoff value for dist.seqs to 0.03. If you must use average neighbor clustering, I’d suggest increasing the cutoff in dist.seqs to 0.15. The modern opticlust algorithm is much improved over average neighbor clustering and shouldn’t have this problem
Pat
Thanks for your advice. After removing method=average, I can assign it to OTU. But I would like to ask what method=average, cutoff=0.03 and label=0.03 mean respectively. Why does label=unique generate ASVs? The official website of mothur describes that ASVs need to set pre.cluster, but it does not clearly explain how to set parameters. Thank you very much for your answer.
Hi - method=average is an old approach that we no longer advocate. i’d suggest reading our OptiClust paper to learn more about the new method.
The MiSeq SOP describes how to generate ASVs using output from pre.cluster directly into make.shared
Pat