Here http://www.mothur.org/wiki/Cluster#hard is said that hard cutoff would leave the cutoff unchanged, but it is still changed to a much lower value. Is there a bug with hard=t option? And is the default value false altough it is said there it is set to true?
The hard cutoff does not stop the cutoff from changing. “Why does the cutoff change” is one of our common questions, here’s Pat’s explanation. “This is a product of using the average neighbor algorithm with a sparse distance matrix. When you run cluster, the algorithm looks for pairs of sequences to merge in the rows and columns that are getting merged together. Let’s say you set the cutoff to 0.05. If one cell has a distance of 0.03 and the cell it is getting merged with has a distance above 0.05 then the cutoff is reset to 0.03, because it’s not possible to merge at a higher level and keep all the data. All of the sequences are still there from multiple phyla. Incidentally, although we always see this, it is a bigger problem for people that include sequences that do not fully overlap.” I would suggest increasing your cutoff.