Technical pitfalls that bias comparative microbial community analyses of aquatic disease Ian Hewson.

The accessibility of high-throughput DNA sequencing technologies has attracted the application of comparative microbial analyses to study diseases. These studies present a window into host microbiome diversity and composition that can be used to address ecological theory in the context of host biology and behavior. Recently, comparative microbiome studies have been used to study non-vertebrate aquatic diseases to elucidate microorganisms potentially involved in disease processes or in disease prevention. These investigations suffer from many well-described biases, especially prior to sequence analyses, that could lead to misleading conclusions. Microbiome-focused studies of aquatic metazoan diseases provide valuable documentation of microbial ecology, although, they are only a starting point for establishing disease etiology, which demands quantitative validation through targeted approaches. The microbiome approach to understanding disease is most useful after laboratory diagnostics guided by pathology have failed to identify a causative agent. This opinion piece presents several technical pitfalls which may affect wider interpretation of microbe-host interactions through comparative microbial community analyses and provides recommendations, based on studies in non-aquatic systems, for incorporation into future aquatic disease research.