Putting COI Metabarcoding in Context: The Utility of Exact Sequence Variants (ESVs) in Biodiversity Analysis

Abstract

DNA barcoding and metabarcoding are techniques that focus on signature genomic regions that in theory provide species level resolution, but in practice this is not always possible. We place animal-focused COI metabarcoding in context with respect to the use of marker gene sequencing in microbial and fungal ecology. We focus on three specific aspects of metabarcodes: (1) the process of metabarcode sequence clustering, (2) how metabarcode cluster types affect the results of biodiversity analyses, and (3) the current state of reference sequence databases used for metabarcode identification. Using examples from the arthropod COI metabarcode literature, we show that exact sequence variants (ESVs) detect more unique taxa than operational taxonomic units (OTUs) but with similar patterns in taxonomic resolution. We also show that the difference between ordinations based on ESVs or OTUs recover similar groupings. We compile a list of reference sequence databases useful for multi-marker metabarcoding and present a list of reference sequence databases specifically formatted for use with a naive Bayesian classifier for rigorous metabarcode taxonomic assignments. Sophisticated tools and reference databases are available for analyzing COI sequences, and these compare favorably with those available for other metabarcode markers such as the ribosomal RNA genes used to target microbes and fungi.