• Evaluating Established Methods for Rumen 16S rRNA Amplicon Sequencing With Mock Microbial Populations

      McGovern, Emily; Waters, Sinead M.; Blackshields, Gordon; McCabe, Matthew; FACCE-JPI; Teagasc Walsh Fellowship Programme; 2014231 (Frontiers, 2018-06-25)
      The rumen microbiome scientific community has utilized amplicon sequencing as an aid in identifying potential community compositional trends that could be used as an estimation of various production and performance traits including methane emission, animal protein production efficiency, and ruminant health status. In order to translate rumen microbiome studies into executable application, there is a need for experimental and analytical concordance within the community. The objective of this study was to assess these factors in relation to selected currently established methods for 16S phylogenetic community analysis on a microbial community standard (MC) and a DNA standard (DS; ZymoBIOMICSTM). DNA was extracted from MC using the RBBC method commonly used for microbial DNA extraction from rumen digesta samples. 16S rRNA amplicon libraries were generated for the MC and DS using primers routinely used for rumen bacterial and archaeal community analysis. The primers targeted the V4 and V3–V4 region of the 16S rRNA gene and samples were subjected to both 20 and 28 polymerase chain reaction (PCR) cycles under identical cycle conditions. Sequencing was conducted using the Illumina MiSeq platform. As the bacteria contained in the microbial mock community were well-classified species, and for ease of explanation, we used the results of the Basic Local Alignment Search Tool classification to assess the DNA, PCR cycle number, and primer type. Sequence classification methodology was assessed independently. Spearman’s correlation analysis indicated that utilizing the repeated bead beating and column method for DNA extraction in combination with primers targeting the 16S rRNA gene using 20 first-round PCR cycles was sufficient for amplicon sequencing to generate a relatively accurate depiction of the bacterial communities present in rumen samples. These results also emphasize the requirement to develop and utilize positive mock community controls for all rumen microbiomic studies in order to discern errors which may arise at any step during a next-generation sequencing protocol.
    • The Structural and Functional Capacity of Ruminal and Cecal Microbiota in Growing Cattle Was Unaffected by Dietary Supplementation of Linseed Oil and Nitrate

      Popova, Milka; McGovern, Emily; McCabe, Matthew; Martin, Cécile; Doreau, Michel; Arbre, Marie; Meale, Sarah J.; Morgavi, Diego P.; Waters, Sinead M.; INRA; et al. (Frontiers, 2017-05-24)
      Microorganisms in the digestive tract of ruminants differ in their functionality and ability to use feed constituents. While cecal microbiota play an important role in post-rumen fermentation of residual substrates undigested in the rumen, limited knowledge exists regarding its structure and function. In this trial we investigated the effect of dietary supplementation with linseed oil and nitrate on methane emissions and on the structure of ruminal and cecal microbiota of growing bulls. Animals were allocated to either a CTL (control) or LINNIT (CTL supplemented with 1.9% linseed and 1.0% nitrates) diet. Methane emissions were measured using the GreenFeed system. Microbial diversity was assessed using amplicon sequencing of microbial genomic DNA. Additionally, total RNA was extracted from ruminal contents and functional mcrA and mtt genes were targeted in amplicon sequencing approach to explore the diversity of functional gene expression in methanogens. LINNIT had no effect on methane yield (g/kg DMI) even though it decreased methane production by 9% (g/day; P < 0.05). Methanobrevibacter- and Methanomassiliicoccaceae-related OTUs were more abundant in cecum (72 and 24%) compared to rumen (60 and 11%) irrespective of the diet (P < 0.05). Feeding LINNIT reduced the relative abundance of Methanomassiliicoccaceae mcrA cDNA reads in the rumen. Principal component analysis revealed significant differences in taxonomic composition and abundance of bacterial communities between rumen and cecum. Treatment decreased the relative abundance of a few Ruminococcaceae genera, without affecting global bacterial community structure. Our research confirms a high level of heterogeneity in species composition of microbial consortia in the main gastrointestinal compartments where feed is fermented in ruminants. There was a parallel between the lack of effect of LINNIT on ruminal and cecal microbial community structure and functions on one side and methane emission changes on the other. These results suggest that the sequencing strategy used here to study microbial diversity and function accurately reflected the absence of effect on methane phenotypes in bulls treated with linseed plus nitrate.