The Organelle in the Room: Under-annotated Mitochondrial Reads Bias Coral Microbiome Analysis

Abstract

The microbiomes of tropical corals are actively studied using 16S rRNA gene amplicons to understand microbial roles in coral health, metabolism, and disease resistance. However, primers targeting bacterial and archaeal 16S rRNA genes may also amplify organelle rRNA genes from the coral, associated microbial eukaryotes, and encrusting organisms. In this manuscript, we demonstrate that standard workflows for annotating microbial taxonomy severely under-annotate mitochondrial sequences in 1272 coral microbiomes from the Earth Microbiome Project. This issue prevents annotation of >95% of reads in some samples and persists when using either Greengenes or SILVA taxonomies. Worse, mitochondrial under-annotation varies between species and across anatomy, biasing comparisons of α- and β-diversity. By supplementing existing taxonomies with diverse mitochondrial rRNA sequences, we resolve ~97% of unique unclassified sequences as mitochondrial, without increasing misannotation in mock communities. We recommend using these extended taxonomies for coral microbiome analysis and encourage vigilance regarding similar issues in other hosts.