Comparisons of 16S amplicon versus shallow shotgun metagenomic sequencing
We next sought to determine whether 16S amplicon and shallow shotgun (iGenomx Riptide prepared) sequencing approaches would yield similar descriptions of 13 fecal microbiome samples of Sable Island horses. Shannon index estimates of ASV (amplicon) and species (shotgun metagenomic) diversity derived from these sequencing methods were correlated (R2 = 0.46, t = 3.375,p < 0.01), but shotgun metagenomic estimates of Shannon diversity (6.78 ± 0.34 SE) were consistently higher than amplicon-based estimates (5.78 ± 0.19 SE), even after rarefying datasets to the same sequencing depth (35,000 bacterial read-pairs). Despite correlations in alpha diversity, ASV-level Bray-Curtis distances calculated from amplicon data were not correlated with species-level Bray-Curtis distances in the shotgun metagenomic dataset (mantel test: r = 0.06, p = 0.38). However, the correlation between beta-diversity matrices increased as reads were grouped into successively coarser taxonomic bins: genus (mantel test: r = 0.46, p < 0.01), family (mantel test: r = 0.60, p < 0.01), order (mantel test: r = 0.81, p < 0.01), class (mantel test: r = 0.80, p < 0.01), and phylum (mantel test: r = 0.80, p < 0.01).
To characterize taxa which may be overrepresented or underrepresented in amplicon versus shotgun metagenomic sequence datasets, we compared bacterial family average relative abundance estimates. A total of 73% of reads could be assigned to one of 58 families in the amplicon dataset, while 70% of reads could be assigned to one of 277 families in the shotgun metagenomic dataset. Of these, 53 were observed in both datasets. Amplicon and shotgun metagenomic-based estimates of average (log-transformed) relative abundance were positively correlated (R2 = 0.33, t = 5.174, p< 0.01; Figure 4A). Highly abundant families tended to be overrepresented within the amplicon dataset compared to the shotgun dataset, with the exception of Fibrobacteraceae . Conversely, many families which were present at moderate relative abundances in the shotgun metagenomic dataset, were either absent or present at lower relative abundances in the amplicon dataset. However, despite the departure of Families from a 1:1 relationship, amplicon and shotgun based estimate of relative abundance were strongly correlated among families previously identified as dominant in the Sable Island horse microbiome: Ruminococcaceae (R2 = 0.49,t = 3.535, p < 0.01), Lachnospiraceae(R2 = 0.55, t = 3.954, p< 0.01), Prevotellaceae (R2 = 0.73, t = 5.753, p < 0.01),Fibrobacteraceae (R2 = 0.91, t = 10.806, p < 0.01), Spirochaetaceae(R2 = 0.84, t = 7.863, p< 0.01), and Rikenellaceae (R2= 0.76, t = 6.314, p < 0.01; Figure 4B).