Best practice for wildlife gut microbiome research: A comprehensive review of methodology for 16S rRNA gene investigations

Extensive research in well-studied animal models underscores the importance of commensal gastrointestinal (gut) microbes to animal physiology. Gut microbes have been shown to impact dietary digestion, mediate infection, and even modify behavior and cognition. Given the large physiological and pathophysiological contribution microbes provide their host, it is reasonable to assume that the vertebrate gut microbiome may also impact the fitness, health and ecology of wildlife. In accordance with this expectation, an increasing number of investigations have considered the role of the gut microbiome in wildlife ecology, health, and conservation. To help promote the development of this nascent field, we need to dissolve the technical barriers prohibitive to performing wildlife microbiome research. The present review discusses the 16S rRNA gene microbiome research landscape, clarifying best practices in microbiome data generation and analysis, with particular emphasis on unique situations that arise during wildlife investigations. Special consideration is given to topics relevant for microbiome wildlife research from sample collection to molecular techniques for data generation, to data analysis strategies. Our hope is that this article not only calls for greater integration of microbiome analyses into wildlife ecology and health studies but provides researchers with the technical framework needed to successfully conduct such investigations.