Re-examination of a published sample-set using shallow shotgun metagenomics
Amplicon and shotgun metagenomic sequencing painted similar portraits of the Sable Island horse microbiome, but we sought to determine whether shallow shotgun sequencing would recapitulate results first described in an amplicon dataset . A primary finding in this amplicon dataset—a clear beta-diversity by spatial distance relationship—was present (but weaker) in the shallow shotgun dataset. Although partial-length 16S amplicon reads cannot reliably be assigned to species, and frequently not to genus, grouping reads to ASVs can still provide a high-resolution picture of the microbiome. In fact, this approach can sometimes be too sensitive, separating 16S rRNA gene reads derived from the same bacterial genome, into separate ASV bins By contrast, the read-based classification of shotgun reads is constrained by taxonomic demarcations recorded in reference databases. Therefore, amplicon sequencing—or deep shotgun sequencing which allows for de novo MAG assembly and strain-resolved tracking—might be better suited than shallow shotgun sequencing for characterizing relationships in the microbiome which are mechanistically driven by microbiota dispersal.
The assembly of Sable Island horse specific MAGs could help increase the resolution of shallow shotgun read classification, however, increased taxonomic resolution is not always useful, since it can obscure biological patterns or cause spurious correlations if explanatory variables are spatially structured; like sea sandwort on Sable Island .
We observed that horse access to sea sandwort was correlated with microbiome beta-diversity differences in both the amplicon and shallow shotgun metagenomic datasets. Although the taxa identified by differential abundance were not identical, both broadly support the same biological conclusion. Namely, that marram grass better supports fibrolytic niches in the microbiome than sea sandwort. In the 16S amplicon dataset, plant fibre specialist bacteria were more abundant when sandwort was absent (Fibrobacteraceae , ;Spirochaetaceae , ), while simple sugar fermenters were more abundant when horses had access to sea sandwort (Christensenellaceae , ). Similarly, in the shallow shotgun metagenomic dataset, plant fibre specialists were more abundant when sandwort was absent (Fibrobacteraceae and a marginally non-significant trend in Spirochaetaceae ) while a functionally diverse clade of plant, host, and alpha glycan foragers,Muribaculaceae , were more abundant when sea sandwort was present.
Interestingly, sea sandwort was associated with bacterial families which contain members capable of degrading the plant toxin oxalate, in both the 16S amplicon (Burkholderiaceae , Hervé, Junier, Bindschedler, Verrecchia, & Junier, 1982), and shallow shotgun datasets (Muribaculaceae , ; Oxalobacteraceae , ). Despite discrepancies in the taxa implicated, data from both sequencing methods suggest sea sandwort might be a source of oxalate in the Sable Island horse diet. Conventional analysis of 16S rRNA gene amplicon data would stop at this point; using taxon differential abundance results to make ecological inferences.