Total rRNA-Seq Analysis Gives Insight into Bacterial, Fungal, Protozoal and Archaeal Communities in the Rumen Using an Optimized RNA Isolation Method

Abstract

Advances in high throughput, next generation sequencing technologies have allowed an in-depth examination of biological environments and phenomena, and are particularly useful for culture-independent microbial community studies. Recently the use of RNA for metatranscriptomic studies has been used to elucidate the role of active microbes in the environment. Extraction of RNA of appropriate quality is critical in these experiments and TRIzol reagent is often used for maintaining stability of RNA molecules during extraction. However, for studies using rumen content there is no consensus on (1) the amount of rumen digesta to use or (2) the amount of TRIzol reagent to be used in RNA extraction procedures. This study evaluated the effect of using various quantities of ground rumen digesta and of TRIzol reagent on the yield and quality of extracted RNA. It also investigated the possibility of using lower masses of solid-phase rumen digesta and lower amounts of TRIzol reagent than is used currently, for extraction of RNA for metatranscriptomic studies. We found that high quality RNA could be isolated from 2 g of ground rumen digesta sample, whilst using 0.6 g of ground matter for RNA extraction and using 3 mL (a 5:1 TRIzol : extraction mass ratio) of TRIzol reagent. This represents a significant savings in the cost of RNA isolation. These lower masses and volumes were then applied in the RNA-Seq analysis of solid-phase rumen samples obtained from 6 Angus X Hereford beef heifers which had been fed a high forage diet (comprised of barley straw in a forage-to-concentrate ratio of 70:30) for 102 days. A bioinformatics analysis pipeline was developed in-house that generated relative abundance values of archaea, protozoa, fungi and bacteria in the rumen and also allowed the extraction of individual rRNA variable regions that could be analyzed in downstream molecular ecology programs. The average relative abundances of rRNA transcripts of archaea, bacteria, protozoa and fungi in our samples were 1.4 ± 0.06, 44.16 ± 1.55, 35.38 ± 1.64, and 16.37 ± 0.65% respectively. This represents the first study to define the relative active contributions of these populations to the rumen ecosystem and is especially important in defining the role of the anaerobic fungi and protozoa.

PCR and Omics Based Techniques to Study the Diversity, Ecology and Biology of Anaerobic Fungi: Insights, Challenges and Opportunities

“…Recently, three studies used sequencing of total RNA, not depleted of rRNA, to examine the active microbial community in the rumen (Poulsen et al, 2013 ; Li F., et al, 2016 ; Elekwachi et al, 2017 ). However, only the study of Elekwachi et al ( 2017 ) found significant contributions of anaerobic fungi.…”

Dynamic role of single‐celled fungi in ruminal microbial ecology and activities

“…Rumen fungi (10 3 -10 6 zoospores per ml) are anaerobic, and also represent approximately 10-20% of the rumen microbiome (Elekwachi et al 2017), belongs to the class Neocallimastigomycetes, consisting of six previously recognized genera (Anaeromyces, Caecomyces, Cyllamyces, Neocallimastix, Orpinomyces and Piromyces; Table 3). Rumen fungi have amylolytic (Gordon and Phillips 1998) and proteolytic activities (Gruninger et al 2014).…”

Characterization of Anaerobic Rumen Fungal Community Composition in Yak, Tibetan Sheep and Small Tail Han Sheep Grazing on the Qinghai-Tibetan Plateau

“…Only 29 species of anaerobic fungi have been identified using culture-dependent techniques [4]. Anaerobic fungi, despite their low percentage (10-20%) of ruminal microbes based on rRNA transcript abundance [6], play a vital role in fiber degradation, primarily due to their efficiency and their producing of an extensive set of polysaccharide-degrading enzymes for plant materials degradation [7,8]. Moreover, rumen fungi have amylolytic and proteolytic activity [9].…”