Microbial communities in guts flexibly adjust to changes in host dietary intakes, but the relationship between diet and gut microbiome is still poorly studied in wild animals. DNA metabarcoding approaches are frequently used to characterise diets or gut microbiomes of diverse species. However, to date, no study has combined these approaches to investigate diet-gut microbiome associations in wild mammals with diverse and fluctuating dietary intakes, such as omnivores. Here, we do this for two African mammals, Civettictis civetta and Genetta spp., from the family Viverridae. We characterised bacterial communities and identified taxonomic groups within diet by sequencing vertebrate, invertebrate and plant markers on faecal samples. This led us to establish diet compositions that diverged from what has previously been found using visual identification methods for these species. Specifically, while the two genera have been categorised into the same dietary guild, we detected more animal-based diets in C. Civetta, and higher proportions of plants consumed by Genetta spp. Diet similarity correlated with gut microbiome similarity in Genetta spp., indicating that plant consumption may be an important driver of gut microbiome structure. The novel insights we provide into the omnivorous diet of C. civetta and Genetta spp. highlight the importance of detailed identification of the dietary guild of species, not only for ecology and conservation, but also when researching how diet shapes the gut microbiome.

Metabarcoding of DNA extracted from environmental or bulk specimen samples is increasingly used to detect plant and animal taxa in basic and applied biodiversity research because of its targeted nature that allows sequencing of genetic markers from many samples in parallel. To achieve this, PCR amplification is carried out with primers designed to target a taxonomically informative marker within a taxonomic group, and sample-specific nucleotide identifiers are added to the amplicons prior to sequencing. This enables assignment of the sequences back to the samples they originated from. Nucleotide identifiers can be added during the metabarcoding PCR and/or during ‘library preparation’, i.e. when amplicons are prepared for sequencing. Different strategies to achieve this labelling exist. All have advantages, challenges and limitations, some of which can lead to misleading results, and in the worst case compromise the fidelity of the metabarcoding data. Given the range of questions addressed using metabarcoding, the importance of ensuring that data generation is robust and fit for purpose should be at the forefront of practitioners seeking to employ metabarcoding for biodiversity assessments. Here, we present an overview of the three main workflows for sample-specific labelling and library preparation in metabarcoding studies on Illumina sequencing platforms. Further, we distil the key considerations for researchers seeking to select an appropriate metabarcoding strategy for their specific study. Ultimately, by gaining insights into the consequences of different metabarcoding workflows, we hope to further consolidate the power of metabarcoding as a tool to assess biodiversity across a range of applications.