Prevailing 16S rRNA gene-amplicon methods for characterizing the bacterial microbiome of wildlife are economical, but result in coarse taxonomic classifications, are subject to primer and 16S copy number biases, and do not allow for direct estimation of microbiome functional potential. While deep shotgun metagenomic sequencing can overcome many of these limitations, it is prohibitively expensive for large sample sets. We evaluated the ability of shallow shotgun metagenomic sequencing to characterize taxonomic and functional patterns in the fecal microbiome of a model population of feral horses (Sable Island, Canada). Since 2007, this unmanaged population has been the subject of an individual-based, long-term ecological study. Using deep shotgun metagenomic sequencing, we determined the sequencing depth required to accurately characterize the horse microbiome. In comparing conventional versus high-throughput shotgun metagenomic library preparation techniques, we validate the use of more cost-effective lab methods. Finally, we characterize similarities between 16S amplicon and shallow shotgun characterization of the microbiome and demonstrate that the latter recapitulates biological patterns first described in a published amplicon dataset. Unlike amplicon data, we further demonstrate how shallow shotgun metagenomic data provide useful insights about microbiome functional potential which support previously hypothesized diet effects in this study system.

16S amplicon, deep shotgun metagenomic, and shallow shotgun metagenomic seqeunce characterization of the bacterial microbiome of feral horse fecal samples. Fresh, individual-linked fecal samples were collected from Sable Island feral horses (Sable Island, Nova Scotia) in 2014, during annual surveys of the horse population. Samples were kept on ice in the field and stored at -20C on Sable Island for two months, before being placed into longterm storage at -80C on the mainland.