Mining for the rumen rare biosphere : a thesis presented in partial fulfilment of the requirement for the degree of Masters in Microbiology, Massey University, Manawatu, New Zealand
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Date
2020
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Massey University
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Abstract
The microbial diversity present in the gut microbiome of ruminant animals is of great interest due to its effect on the New Zealand economy. The rumen, a forestomach of ruminants, is a large fermentation chamber. The microbiome within the rumen influences production of milk and meat, and additionally impacts on climate change through the emission of enteric methane. Although, the core microbiome has been studied intensely, the rare biosphere, which is comprised of the rare microorganisms present in less than 0.1% of the abundance, is still largely unknown. Recent developments in methods for subtraction, or normalisation, of the dominant microorganisms from analysis of complex microbiomes, including treatment with duplex-specific nuclease (DSN), have enabled the increase of the number of sequences from low abundance microorganisms. Decreasing presence of dominant species and simultaneously increasing low abundant allows the exploration of the rare biosphere and discovery of taxa which otherwise would not have been identified. By applying DSN-based normalisation to a metagenomic DNA isolated from the rumen microbiome, we have demonstrated that the low abundance microorganisms, can be amplified to a detectable level while decreasing the abundance of sequences from dominant species. The outcome of DNA normalisation, primarily taxonomic assignment and phylogeny was assessed by using the gene encoding the β subunit of bacterial RNA polymerase, rpoB, as well as the “gold standard” 16S rRNA as phylogenetic markers. We have demonstrated that rpoB could be effectively used for determining the rumen microbial community profile and could become by broader adoption from researchers, a valuable resource for microbial ecology studies. We suggest that DSN-based normalisation could be utilised for in-depth exploration of the rare biosphere as a whole, resulting in the discovery of new species, new genes and increasing understanding of the role that these rare microorganisms play in the rumen microbiome. The inclusion of rpoB, alone or in combination with 16S rRNA marker, in microbial ecology studies could lead to more accurate classification of the taxa.