Accessing Dietary Effects on the Rumen Microbiome: Different Sequencing Methods Tell Different Stories

Abstract

The current study employed both amplicon and shotgun sequencing to examine and compare the rumen microbiome in Angus bulls fed with either a backgrounding diet (BCK) or finishing diet (HG), to assess if both methods produce comparable results. Rumen digesta samples from 16 bulls were subjected for microbial profiling. Distinctive microbial profiles were revealed by the two methods, indicating that choice of sequencing approach may be a critical facet in studies of the rumen microbiome. Shotgun-sequencing identified the presence of 303 bacterial genera and 171 archaeal species, several of which exhibited differential abundance. Amplicon-sequencing identified 48 bacterial genera, 4 archaeal species, and 9 protozoal species. Among them, 20 bacterial genera and 5 protozoal species were differentially abundant between the two diets. Overall, amplicon-sequencing showed a more drastic diet-derived effect on the ruminal microbial profile compared to shotgun-sequencing. While both methods detected dietary differences at various taxonomic levels, few consistent patterns were evident. Opposite results were seen for the phyla Firmicutes and Bacteroidetes, and the genus Selenomonas. This study showcases the importance of sequencing platform choice and suggests a need for integrative methods that allow robust comparisons of microbial data drawn from various omic approaches, allowing for comprehensive comparisons across studies.

Keywords: amplicon-sequencing; bull cattle; diets; rumen; shotgun-sequencing.

Figure 1

Microbial profiles at different phylogeny levels observed by shotgun-seq. (A) Bacterial phyla; (B) Bacterial families; (C) Bacterial genera; (D) Archaeal species.

Figure 2

Microbial profiles at different phylogeny levels observed by amplicon-seq. (A) Bacterial phyla; (B) Bacterial families; (C) Bacterial genera; (D) Archaeal species; (E) Protozoal species.

Figure 3

Alpha diversity (Chao 1) and beta diversity (Shannon) indices of bacterial, archaeal, and protozoal communities by shotgun-seq and amplicon-seq.

Figure 4

Comparison of the identified phylotypes by shotgun-seq and amplicon-seq. (A–D) Venn diagrams of the bacterial phyla, bacterial families, bacterial species, and archaeal species. (a–d) Relative abundance of the shared phylotypes of the bacterial phyla, bacterial families, bacterial species, and archaeal species.

Figure 5

Comparison of microbial profiles observed by shotgun-seq. (A) PCoA plot of bacterial and archaeal communities. (B) Alpha (Chao 1) and beta (Shannon) indices of bacterial and archaeal communities. (C) Differential abundant phylotypes observed between the two diets. Significance was indicated as * fdr < 0.05 and ** fdr < 0.01.

Figure 6

Comparison of microbial profiles observed by shotgun-seq. (A) PCoA plot of bacterial, archaeal, and protozoal communities. (B) Alpha (Chao 1) and beta (Shannon) indices of bacterial, archaeal, and protozoal communities. (C) Differential abundant phylotypes observed between the two diets. Significance was indicated as * fdr < 0.05 and ** fdr < 0.01.