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).