Comparative analyses of the bovine rumen microbiota using RNA and targeted DNA-based sequencing approaches

Abstract Text: The bovine rumen microbiota comprises diverse populations, including bacteria, archaea, protozoa, and fungi. Defining rumen microbiota structures and their activities can enhance our understanding of the role of microbes in regulating rumen fermentation. We hypothesized that sequencing total RNA without rRNA removal (RNA-Seq) can be used to study the active microbiota, avoiding potential biases introduced by PCR-amplification of bacterial and archaeal 16S rRNA genes prior to sequencing. Total DNA and total RNA were isolated from the rumen contents of five steers maintained on a feedlot diet. Partial amplicons of bacterial and archaeal 16S rRNA genes were sequenced (Amplicon-Seq). RNA-Seq was performed in parallel. The data were processed using a QIIME-based pipeline in combination with Greengenes and SILVA-derived taxonomic frameworks. In total, five major bacterial phyla, with a relative abundance greater than 0.1% in any one sample, were identified in both Amplicon-Seq and RNA-Seq datasets; namely Proteobacteria, Bacteroidetes, Firmicutes, Spirochaetes, and Synergistetes. Bacteroidetes was more abundant in Amplicon-Seq than RNA-Seq datasets (52.6±8.8% versus 23.7±7.7%, mean±SEM), whereas Proteobacteria was predominant in RNA-Seq datasets (45.7±14.1% versus 13.0±7.7%). Euryarchaeota was the most abundant archaeal phylum in both Amplicon-Seq (100.0±0.0%) and RNA-Seq (94.2±2.5%) datasets. Despite some differences in individual animals, microbial community compositions obtained using Amplicon-Seq and RNA-Seq techniques were broadly comparable. Of particular interest is that Proteobacteria appear to be more abundant in RNA-Seq datasets. Whether they are indeed more active relative to their abundance (Amplicon-Seq) than other bacteria warrants further investigation. Our results suggest that total RNA sequencing without rRNA removal can be used to study the active rumen microbiota.

Keywords: Amplicon-Seq, RNA-Seq and Rumen microbiota