An operational taxonomic unit (OTU) is a pragmatic definition used to group closely related organisms or unknown microbes based on DNA sequence similarity. OTUs cluster sequences of a marker gene, like 16S rRNA for prokaryotes, according to a similarity threshold chosen by the researcher, usually 97%. This operational definition allows the study and classification of microbes lacking traditional taxonomy and serves as a proxy for "species" when analyzing sequence datasets.

1. Operational taxonomic unit
• An operational taxonomic unit (OTU) is an operational definition used to
classify groups of closely related individuals.
• The term was originally introduced by Robert R. Sokal and Peter H. A. Sneath in
the context of numerical taxonomy, where an "Operational Taxonomic Unit" is
simply the group of organisms currently being studied.
• In this sense, an OTU is a pragmatic definition to group individuals by similarity,
equivalent to but not necessarily in line with classical Linnaean taxonomy or
modern evolutionary taxonomy.
• Nowadays, however, the term "OTU" is also used in a different context and
refers to clusters of (uncultivated or unknown) organisms, grouped by DNA
sequence similarity of a specific taxonomic marker gene (originally coined as
mOTU; molecular OTU).
• In other words, OTUs are pragmatic proxies for "species" (microbial or
metazoan) at different taxonomic levels, in the absence of traditional systems
of biological classification as are available for macroscopic organisms.
• For several years, OTUs have been the most commonly used units of diversity,
especially when analysing small subunit 16S (for prokaryotes) or 18S rRNA (for
eukaryotes) marker gene sequence datasets.

2. • Sequences can be clustered according to their similarity to one another, and
operational taxonomic units are defined based on the similarity threshold
(usually 97% similarity; however also 100% similarity is common, also known
as single variants) set by the researcher.
• It remains debatable how well this commonly-used method recapitulates true
microbial species phylogeny or ecology.
• Although OTUs can be calculated differently when using different algorithms or
thresholds, recent research by Schmidt, demonstrated that microbial OTUs
were generally ecologically consistent across habitats and several OTU
clustering approaches.
• The number of OTUs defined may be inflated due to errors in DNA sequencing.