ASV and OTU same number of sequences

[jmacnico]

Hi all!!

So I ran both an ASV approach and OTU approach for my analysis but at the count seqs step I have the same number of sequences in all my samples I thought that I should likely have more sequences when I ran my analysis with the ASV approach… just wondering if I might be running something wrong or whether this is possible.

Below are the commands/output. I didn’t include the counts per sequence because we have 300+ samples in this dataset.

OTU approach
mothur > cluster.split(fasta=stability.trim.contigs.good.unique.good.filter.unique.precluster.denovo.vsearch.pick.fasta, count=stability.trim.contigs.good.unique.good.filter.unique.precluster.denovo.vsearch.pick.count_table, taxonomy=stability.trim.contigs.good.unique.good.filter.unique.precluster.denovo.vsearch.pds.wang.pick.taxonomy, taxlevel=4, cutoff=0.03, processors=24)

Output File Names:
stability.trim.contigs.good.unique.good.filter.unique.precluster.denovo.vsearch.pick.dist
stability.trim.contigs.good.unique.good.filter.unique.precluster.denovo.vsearch.pick.opti_mcc.list
stability.trim.contigs.good.unique.good.filter.unique.precluster.denovo.vsearch.pick.opti_mcc.sensspec

mothur > make.shared(list=stability.trim.contigs.good.unique.good.filter.unique.precluster.denovo.vsearch.pick.opti_mcc.list, count=stability.trim.contigs.good.unique.good.filter.unique.precluster.denovo.vsearch.pick.count_table, label=0.03)

Output File Names:
stability.trim.contigs.good.unique.good.filter.unique.precluster.denovo.vsearch.pick.opti_mcc.shared

mothur > classify.otu(list=stability.trim.contigs.good.unique.good.filter.unique.precluster.denovo.vsearch.pick.opti_mcc.list, count=stability.trim.contigs.good.unique.good.filter.unique.precluster.denovo.vsearch.pick.count_table, taxonomy=stability.trim.contigs.good.unique.good.filter.unique.precluster.denovo.vsearch.pds.wang.pick.taxonomy, label=0.03)

Output File Names:
stability.trim.contigs.good.unique.good.filter.unique.precluster.denovo.vsearch.pick.opti_mcc.0.03.cons.taxonomy
stability.trim.contigs.good.unique.good.filter.unique.precluster.denovo.vsearch.pick.opti_mcc.0.03.cons.tax.summary

mothur > count.groups(shared=stability.trim.contigs.good.unique.good.filter.unique.precluster.denovo.vsearch.pick.opti_mcc.shared)

Size of smallest group: 28919.

Total seqs: 43005220.

Output File Names:
stability.trim.contigs.good.unique.good.filter.unique.precluster.denovo.vsearch.pick.opti_mcc.count.summary

ASV approach

mothur > make.shared(count=current)
Using stability.trim.contigs.good.unique.good.filter.unique.precluster.denovo.vsearch.pick.count_table as input file for the count parameter.
ASV

Output File Names:
stability.trim.contigs.good.unique.good.filter.unique.precluster.denovo.vsearch.pick.asv.list
stability.trim.contigs.good.unique.good.filter.unique.precluster.denovo.vsearch.pick.asv.shared

mothur > classify.otu(list=current, count=current, taxonomy=current, label=ASV)
Using stability.trim.contigs.good.unique.good.filter.unique.precluster.denovo.vsearch.pick.count_table as input file for the count parameter.
Using stability.trim.contigs.good.unique.good.filter.unique.precluster.denovo.vsearch.pick.asv.list as input file for the list parameter.
Using stability.trim.contigs.good.unique.good.filter.unique.precluster.denovo.vsearch.pds.wang.pick.taxonomy as input file for the taxonomy parameter.
ASV

Output File Names:
stability.trim.contigs.good.unique.good.filter.unique.precluster.denovo.vsearch.pick.asv.ASV.cons.taxonomy
stability.trim.contigs.good.unique.good.filter.unique.precluster.denovo.vsearch.pick.asv.ASV.cons.tax.summary

mothur > count.groups(shared=stability.trim.contigs.good.unique.good.filter.unique.precluster.denovo.vsearch.pick.asv.shared)
Using stability.trim.contigs.good.unique.good.filter.unique.precluster.denovo.vsearch.pick.asv.shared as input file for the shared parameter.

Size of smallest group: 28919.

Total seqs: 43005220.

Output File Names:
stability.trim.contigs.good.unique.good.filter.unique.precluster.denovo.vsearch.pick.asv.count.summary

Any suggestions/insight would be much appreciated!!

Many thanks,
JM

[pschloss]

Hi there - I’m a bit confused, do you mean that you have the same number of sequences in each of the 300+ groups or that you have the same number of sequences in the same group after doing ASVs and OTUs?

Group X should have the same number of sequences by ASVs and OTUs, but will be likely different from what you get for Group Y. The sequences used to generate ASVs are the same sequences used to generate OTUs and nothing is removed. I would expect to see a difference in the number of OTUs/ASVs. You could assess this by running summary.single using calc=sobs and rarefy=TRUE

Pat