Functional analysis using shallow shotgun metagenomics
An advantage of shallow shotgun sequencing over amplicon sequencing is that it can allow for the direct estimation of microbiome functional potential. Mirroring patterns in taxonomic profiles, Bray-Curtis dissimilarities of estimated metabolic pathways significantly differed between horses with access to sandwort versus those without access to sandwort (PERMANOVA: F = 1.987, R2 = 0.02, p < 0.01). ANCOM-BC analysis (Table S1C) indicated that horses without access to sandwort had greater relative abundances of pathways related to pyruvate fermentation to acetate and lactate (PWY-7357), thiamine phosphate formation (PWY-7357), thiamine diphosphate salvage (PWY-6897), and L-arginine biosynthesis (ARGSYNBSUB-PWY). Conversely, pathways for sucrose degradation (PWY-7345), alongside a suite of other metabolic pathways were enriched when sandwort was present (Figure 7). Interestingly, four of the metabolic pathways significantly associated with horse access to sandwort were Archaea specific. Post hoc re-analysis of the shallow shotgun dataset in which Archaea were retained alongside bacteria, indicated that the ratio of Archaea to Bacteria was nearly doubled when sandwort was present (1.3% ± 0.3% CI ), compared to when sandwort was absent (0.7% ± 0.04% CI ; t = 7.239,p < 0.01).
In addition to metabolic pathways, reads which mapped to genes linked to 56 MetaCyc ‘reactions’ were more relatively abundant among horses with access to sandwort, while 40 were enriched among horses without access to sandwort (Table S1D). Most notably, reads which mapped to cellulase gene regions were more abundant among horses without access to sandwort (Figure 8A), while reads which mapped to genes for fructan beta-fructosidases (Figure 8B) and alpha-amylases (Figure 8C) synthesis were more abundant when sandwort was present.