Principles underlying stable closed-reference clustering, rarefaction curves, and PCoA based on the Bray-Curtis distance. (a) Principles underlying stable closed-reference clustering at two sampling depths. White circles indicate individual sequences that were included in both the small and the large subsamples, and dark circles indicate sequences that were added only in the large subsample. Diamonds indicate reference sequences. Lines indicate pairs of sequences with distances equal to or smaller than the threshold, which could therefore be linked into a single OTU. Large circles in red or blue indicate OTUs in the small and the large subsamples, respectively. (b) Rarefaction curves generated with closed-reference clustering at five different depths. (c) PCoA based on the Bray-Curtis distance, comparing 60% subsamples with the full datasets using closed reference clustering. All of the subsamples were rarefied to 30,000 sequences per sample to be included in this analysis.