Shallow shotgun metagenomic functional analysis
In contrast to 16S amplicon data, shallow shotgun metagenomic data also provide direct estimates of microbiome functional potential. Horses without access to sandwort had greater relative abundances of reads which mapped to genes related to cellulases, enzymes which break down cellulose (the primary structural component of plant cell walls) into monosaccharides. Horses without access to sandwort were also enriched in metabolic pathways for pyruvate fermentation to acetate (and S-lactate). In horses, acetate is the primary fatty acid produced at the end of a metabolic chain which begins with the lysis of plant fibres . Conversely, metabolic pathways for sucrose and host glycan (N-Acetylneuraminic acid) degradation were enriched among horses with access to sandwort, alongside genes for amylase and fructosidase synthesis. Compared to taxonomic patterns, these functional results more directly support our hypothesis that sea sandwort consumption reduces fibrolytic niche space in the microbiome, and shifts communities towards the metabolism of simple sugars and host glycans.
Contrary to taxonomic indicators, we did not observe an association between oxalate decarboxylase genes and horse access to sandwort. Although reads which mapped to oxalate decarboxylase genes were observed, they were present at low abundances (< 10 reads) and not observed in all samples. This highlights a limitation of shallow shotgun sequencing. While shallow shotgun sequencing may be sufficient to detect patterns in genes which are abundant and widely distributed in the microbiome, genes related to specialized functions which are only found in a rare subset of taxa may require deep shotgun metagenomic sequencing or targeted sequencing.