Re-examination of a published sample-set using shallow shotgun
metagenomics
After establishing that 16S amplicon and shallow shotgun metagenomic
sequencing yielded similar estimates of taxonomic patterns, we sought to
determine whether a shallow shotgun sequencing approach could
recapitulate the findings reported in a recently published 16S rRNA
amplicon sequence dataset .
A primary finding of this paper was an apparent relationship between
microbiome ASV-level beta-diversity and the spatial separation of sample
collection locations. Spatial patterns in the microbiome were still
present in an 83-sample subset of the previously published amplicon
dataset (mantel test: r = 0.51, p < 0.01; Figure
5), even after controlling for sea sandwort availability (partial
mantel test: r = 0.25, p < 0.01), a
heterogeneously distributed dietary component shown to correlate with
microbiome variation. A much weaker spatial effect was observed in the
shallow shotgun metagenomic dataset (mantel test: r = 0.19,p < 0.01), and this effect was only marginally
significant after controlling for sea sandwort availability(partial mantel test: r = 0.12, p = 0.04). Despite an
absence of correlation in ASV/species level beta-diversity in a
13-sample set of re-sequenced DNA extracts (see above), we did observe a
modest correlation in amplicon and shotgun metagenomic estimated
Bray-Curtis dissimilarity in this expanded 83-sample dataset (mantel
test: r = 0.44, p < 0.01). However,
dissimilarity estimates in the amplicon dataset were consistently higher
than those in the shallow shotgun dataset. This could be caused by a
zero-inflated ASV count-table in the amplicon dataset. This pattern is
common in 16S rRNA gene amplicon studies , and was evidenced by an
apparent bi-modal distribution in ASV prevalence (Figure S4).
In addition to spatial effects, we previously demonstrated that horse
access to sandwort appeared to affect the Sable Island horse bacterial
microbiome . This pattern was still evident in a reduced 83-sample
subset of these data (PERMANOVABray-Curtis: F =
5.300, R2 = 0.06, p < 0.01),
and we observed an effect of comparable magnitude within the shallow
shotgun metagenomic dataset (PERMANOVABray-Curtis:F = 6.146, R2 = 0.07, p< 0.01).
Analysis of 16S rRNA amplicon sequence data using ANCOM-BC tests
indicated that Fibrobacteraceae , Spirochaetaceae , andPasteurellaceae were enriched in horses without access to
sandwort, while horses with access to sandwort tended to possess greater
abundances of Christensenellaceae , Anaeroplasmataceae ,Mycoplasmataceae , Peptococcaceae , Burkholderiaceae ,
and Puniceicoccaceae (Table S1A). Within the shallow shotgun
dataset, Fibrobacteraceae and Nanosyncoccacae (candidate
family) were significantly enriched among horses without access to
sandwort, while Oxalobacteraceae and Muribaculaceae were
of greater abundance in horses with access to sandwort (Table S1B).
Despite non-identical patterns in the Families identified between
datasets, there was a positive correlation in the test statistics
(log-linear difference divided by the standard error) among families
present in both datasets (R2 = 0.26, t =
3.717, p < 0.01; Figure 6).