Shallow shotgun sequencing reduces technical variation in microbiome analysis.

The microbiome is known to play a role in many human diseases, but identifying key microbes and their functions generally requires large studies due to the vast number of species and genes, and the high levels of intra-individual and inter-individual variation. 16S amplicon sequencing of the rRNA gene is commonly used for large studies due to its comparatively low sequencing cost, but it has poor taxonomic and functional resolution. Deep shotgun sequencing is a more accurate and comprehensive alternative for small studies, but can be cost-prohibitive for biomarker discovery in large populations. Shallow or moderate-depth shotgun metagenomics may serve as a viable alternative to 16S sequencing for large-scale and/or dense longitudinal studies, but only if resolution and reproducibility are comparable. Here we applied both 16S and shallow shotgun stool microbiome sequencing to a cohort of 5 subjects sampled twice daily and weekly, with technical replication at the DNA extraction and the library preparation/sequencing steps, for a total of 80 16S samples and 80 shallow shotgun sequencing samples. We found that shallow shotgun sequencing produced lower technical variation and higher taxonomic resolution than 16S sequencing, at a much lower cost than deep shotgun sequencing. These findings suggest that shallow shotgun sequencing provides a more specific and more reproducible alternative to 16S sequencing for large-scale microbiome studies where costs prohibit deep shotgun sequencing and where bacterial species are expected to have good coverage in whole-genome reference databases.

Assessing the relationship between the rumen microbiota and feed efficiency in Nellore steers.

BACKGROUND: Ruminants rely upon a complex community of microbes in their rumen to convert host-indigestible feed into nutrients. However, little is known about the association between the rumen microbiota and feed efficiency traits in Nellore (Bos indicus) cattle, a breed of major economic importance to the global beef market. Here, we compare the composition of the bacterial, archaeal and fungal communities in the rumen of Nellore steers with high and low feed efficiency (FE) phenotypes, as measured by residual feed intake (RFI). RESULTS: The Firmicutes to Bacteroidetes ratio was significantly higher (P < 0.05) in positive-RFI steers (p-RFI, low feed efficiency) than in negative-RFI (n-RFI, high feed efficiency) steers. The differences in bacterial composition from steers with high and low FE were mainly associated with members of the families Lachnospiraceae, Ruminococcaceae and Christensenellaceae, as well as the genus Prevotella. Archaeal community richness was lower (P < 0.05) in p-RFI than in n-RFI steers and the genus Methanobrevibacter was either increased or exclusive of p-RFI steers. The fungal genus Buwchfawromyces was more abundant in the rumen solid fraction of n-RFI steers (P < 0.05) and a highly abundant OTU belonging to the genus Piromyces was also increased in the rumen microbiota of high-efficiency steers. However, analysis of rumen fermentation variables and functional predictions indicated similar metabolic outputs for the microbiota of distinct FE groups. CONCLUSIONS: Our results demonstrate that differences in the ruminal microbiota of high and low FE Nellore steers comprise specific taxa from the bacterial, archaeal and fungal communities. Biomarker OTUs belonging to the genus Piromyces were identified in animals showing high feed efficiency, whereas among archaea, Methanobrevibacter was associated with steers classified as p-RFI. The identification of specific RFI-associated microorganisms in Nellore steers could guide further studies targeting the isolation and functional characterization of rumen microbes potentially important for the energy-harvesting efficiency of ruminants.

The Bacterial and Fungal Microbiota of Nelore Steers Is Dynamic Across the Gastrointestinal Tract and Its Fecal-Associated Microbiota Is Correlated to Feed Efficiency.

The ruminant gastrointestinal tract (GIT) microbiome plays a major role in the health, physiology and production traits of the host. In this work, we characterized the bacterial and fungal microbiota of the rumen, small intestine (SI), cecum and feces of 27 Nelore steers using next-generation sequencing and evaluated biochemical parameters within the GIT segments. We found that only the bacterial microbiota clustered according to each GIT segment. Bacterial diversity and richness as well as volatile fatty acid concentration was lowest in the SI. Taxonomic grouping of bacterial operational taxonomic units (OTUs) revealed that Lachnospiraceae (24.61 ± SD 6.58%) and Ruminococcaceae (20.87 ± SD 4.22%) were the two most abundant taxa across the GIT. For the fungi, the family Neocallismastigaceae dominated in all GIT segments, with the genus Orpinomyces being the most abundant. Twenty-eight bacterial and six fungal OTUs were shared across all GIT segments in at least 50% of the steers. We also evaluated if the fecal-associated microbiota of steers showing negative and positive residual feed intake (n-RFI and p-RFI, respectively) was associated with their feed efficiency phenotype. Diversity indices for both bacterial and fungal fecal microbiota did not vary between the two feed efficiency groups. Differences in the fecal bacterial composition between high and low feed efficiency steers were primarily assigned to OTUs belonging to the families Lachnospiraceae and Ruminococcaceae and to the genus Prevotella. The fungal OTUs shared across the GIT did not vary between feed efficiency groups, but 7 and 3 OTUs were found only in steers with positive and negative RFI, respectively. These results provide further insights into the composition of the Nelore GIT microbiota, which could have implications for improving animal health and productivity. Our findings also reveal differences in fecal-associated bacterial OTUs between steers from different feed efficiency groups, suggesting that fecal sampling may represent a non-invasive strategy to link the bovine microbiota with productivity phenotypes.

The Ruminococci: key symbionts of the gut ecosystem.

Mammalian gut microbial communities form intricate mutualisms with their hosts, which have profound implications on overall health. One group of important gut microbial mutualists are bacteria in the genus Ruminococcus, which serve to degrade and convert complex polysaccharides into a variety of nutrients for their hosts. Isolated decades ago from the bovine rumen, ruminococci have since been cultured from other ruminant and non-ruminant sources, and next-generation sequencing has further shown their distribution to be widespread in a diversity of animal hosts. While most ruminococci that have been studied are those capable of degrading cellulose, much less is known about non-cellulolytic, nonruminant-associated species, such as those found in humans. Furthermore, a mechanistic understanding of the role of Ruminococcus spp. in their respective hosts is still a work in progress. This review highlights the broad work done on species within the genus Ruminococcus with respect to their physiology, phylogenetic relatedness, and their potential impact on host health.

Sequence-based analysis of the genus Ruminococcus resolves its phylogeny and reveals strong host association.

It has become increasingly clear that the composition of mammalian gut microbial communities is substantially diet driven. These microbiota form intricate mutualisms with their hosts, which have profound implications on overall health. For example, many gut microbes are involved in the conversion of host-ingested dietary polysaccharides into host-usable nutrients. One group of important gut microbial symbionts are bacteria in the genus Ruminococcus. Originally isolated from the bovine rumen, ruminococci have been found in numerous mammalian hosts, including other ruminants, and non-ruminants such as horses, pigs and humans. All ruminococci require fermentable carbohydrates for growth, and their substrate preferences appear to be based on the diet of their particular host. Most ruminococci that have been studied are those capable of degrading cellulose, much less is known about non-cellulolytic non-ruminant-associated species, and even less is known about the environmental distribution of ruminococci as a whole. Here, we capitalized on the wealth of publicly available 16S rRNA gene sequences, genomes and large-scale microbiota studies to both resolve the phylogenetic placement of described species in the genus Ruminococcus, and further demonstrate that this genus has largely unexplored diversity and a staggering host distribution. We present evidence that ruminococci are predominantly associated with herbivores and omnivores, and our data supports the hypothesis that very few ruminococci are found consistently in non-host-associated environments. This study not only helps to resolve the phylogeny of this important genus, but also provides a framework for understanding its distribution in natural systems.