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.