Principles underlying unstable single linkage (SL) clustering, rarefaction curves, and PCoA based on the Bray-Curtis distance. (a) Principles underlying unstable SL 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 are added only in the large subsample. 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, d) Rarefaction curves generated with SL (b) and average linkage (AL) (d) clustering at five different depths. (c, e) PCoA based on the Bray-Curtis distance, comparing 60% subsamples with the full datasets using SL (c) and AL (e). All of the subsamples were rarefied to 30,000 sequences per sample to be included in this analysis.