cluster.split/ make.shared -> labels?

Dear mothur-forum members,

we are in the process of going through the SOP-protocol provided by Patrick Schloss. At the moment we are a bit confused at the cluster.split and make.shared command, respectively.
As far as we understand the OTU’s are generated by the cluster.split command by a distance matrix calculation using a cutoff value. Which determines which representing sequences are used for each distance matrix. One question would be, what happens to the sequences which have a greater distance then the cutoff-level. Are they pooled in a new OTU?
After cluster.split a …unique_list.list file is generated. When we look inside the file the first column is designated “label”. What does this mean “label”? We see that the numbers of OTU’s is decreasing from unique -> 0.06 and the amount of the representative sequences in the respective OTU’s are increasing.
Then with the make.shared command we select for the labels “unique”, “0.01”, and so on. We are not sure for what we are selecting.
Any comment or hint is highly appreciated!
Thank you very much in advance!
Regards,

Thanks for your questions…

One question would be, what happens to the sequences which have a greater distance then the cutoff-level. Are they pooled in a new OTU?

The pairwise distances that above the cutoff are discarded since those sequences could not appear in the same OTU as each other.

After cluster.split a …unique_list.list file is generated. When we look inside the file the first column is designated “label”. What does this mean “label”? We see that the numbers of OTU’s is decreasing from unique -> 0.06 and the amount of the representative sequences in the respective OTU’s are increasing. Then with the make.shared command we select for the labels “unique”, “0.01”, and so on. We are not sure for what we are selecting.

The label refers to the OTU definition that was used. Typically people are most interested in 0.03. This means that, on average, the sequences within an OTU are no more than 3% different from each other.

Pat

Dear Pat,

thank you for taking the time to answer our questions!
Your clarification helped a lot!

Regards,