OTU variability observed through comprehensive methodology search. (a) The number of OTUs found versus the VI distance from the annotated species clustering for 749 OTU sets. Generally, smaller clustering distances lead to many OTUs while larger clustering distances result in very few OTUs, both of which poorly approximate the species-level structure in the sample. Near 49 OTUs, the true number of species in the simulated sample, the OTU sets are relatively closer to the true species structure. Detail of the lower-left corner of (a) re-colored by (b) MSA, (c) distance threshold, and (d) clustering algorithm.