OTUs

An operational taxonomic unit (OTU) is a pragmatic unit used in microbial ecology to group similar sequences from marker genes, most commonly 16S rRNA genes. OTUs are used as proxies for taxonomic groups in sequencing surveys and are not formal species.

OTUs are created by clustering sequences that exceed a similarity threshold. In de novo OTU clustering, sequences from a dataset are grouped into OTUs based solely on pairwise similarity, typically using a 97% identity threshold for bacteria; thresholds may vary (for example, 95% for coarser groupings). In closed-reference OTU picking, sequences are mapped to a reference database and only those that match form OTUs; open-reference combines both approaches.

Typical pipelines (such as QIIME and mothur) implement OTU clustering and subsequent analyses of community composition and diversity. OTU counts are then used for alpha diversity (within-sample diversity) and beta diversity (between-sample differences), as well as relative abundance summaries and downstream ecological tests.

Limitations include the arbitrary nature of similarity thresholds, susceptibility to sequencing errors and chimeras, and the fact that an OTU may group together several distinct taxa or split a single taxon into multiple OTUs. Cross-study comparability is also hampered by different reference databases and clustering methods.

In recent years, amplicon sequence variants (ASVs) produced by denoising methods such as DADA2 and Deblur have gained prominence because they resolve sequences at single-nucleotide resolution and facilitate cross-study comparisons. Nevertheless, OTUs remain in use, particularly for legacy datasets and certain analyses.