Question on pre.cluster-based ASVs

Hi mothur developers,

In the MiSeq SOP, pre.cluster ASVs are created after having removed chimeras. Shouldn’t chimeras be removed before the pre.cluster step?

Thanks very much.

In the SOP pre.cluster is run before removing chimeras and tossing contaminants. If you look at the syntax for building the shared file you’ll see…

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

That count file has precluster.denovo.vsearch in its name, which tells you that it’s the count file from after removing chimeras. There are also three pick's in the file name. Picks tell you sequences were removed. In this case the chimeras, contaminants, and mock sequences were removed.

Hi Pat,

Thank you for the quick reply. I now realize I posed the question incorrectly; I had in fact noticed that the order of the commands indicates that pre.cluster is run before removing chimeras. My question is: for creating ASVs using the pre.clustermake.shared command sequence, shouldn’t chimera.vsearch be run BEFORE pre.cluster? If not, how does one compare ASV vs. OTU error rates?

Thanks again.`

The same file that is used for making OTUs is used for making ASVs. It’s the same count file for the same fasta file. You need to run pre.cluster before chimera checking so that you have the best representation of the frequency of the sequences.


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