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ABSTRACT Assignment of 16S rRNA gene sequences to operational taxonomic units (OTUs) allows microbial ecologists to overcome the inconsistencies and biases within bacterial taxonomy and provides a strategy for clustering similar sequences that do not have representatives in a reference database. I have applied the Matthews correlation coefficient to assess the ability of 15 reference-independent and -dependent clustering algorithms to assign sequences to OTUs. This metric quantifies the ability of an algorithm to reflect the relationships between sequences without the use of a reference and can be applied to any data set or method. The most consistently robust method was the average neighbor algorithm; however, for some data sets, other algorithms matched its performance.