OTUs contain contigs that are 97% similar

Commands in mothur
1

Dear mothur team,

I’ve started to use mothur some month ago to analyse V4-s of 16SrRNAs of gut microbiomes based on the MiSeq SOP. Although, the user’s guide of mothur is excellent, I’ve got confused about how to set the cutoff parameter of cluster.split and the label parameter of make.shared and classify.otu commands if I would like to have clusters contain contigs that are 97% similar and – in another run – 100% identical.
Previously I’ve set the cutoff to 0.015 based on the explanation I’ve found here: http://www.mothur.org/wiki/Cluster.split#cutoff. But lately I saw another suggestion here: http://www.mothur.org/wiki/MiSeq_SOP#OTUs (cutoff=0.15).
Could you help me to understand these parameters and decide how to set up my pipeline?

Thanks a lot!
István

2

You want to use 0.15 - http://www.mothur.org/wiki/Frequently_asked_questions#Why_does_the_cutoff_change_when_I_cluster_with_average_neighbor.3F

Pat

3

Dear Pat!
Thank you for your answer.

So, based on the FAQ and other forum topic, I ran the cluster.split command with cutoff=0.15 resulted the .list file with labels: unique, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06.

Then I ran the make.shared, classify.otu, get.sharedseqs, get.otulist and get.oturep commands with parameter label=0.03 for clusters contain contigs that are 97% similar.

And with parameter label=unique for clusters contain contigs that are 100% similar.

Is that correct?

Thanks, Istvan

4

correct.

5

Thanks a lot, Pat!