Low-cost sample preservation methods for high-throughput processing of rumen microbiomes

The use of ruminal microbiome profiles to predict methane emissions has driven interest in ruminal DNA preservation and extraction protocols that can be automated cheaply while also maintaining or improving DNA quality for microbial profiling. The standard approach for preserving rumen samples as defined in the Global Rumen Census (GRC) requires time-consuming pre-processing steps of freeze drying and grinding prior to international transportation and DNA extraction. To circumvent these pre-processing steps, we investigated three methods of preserving rumen samples for subsequent DNA extraction, based on existing lysis buffers Tris-NaCl-EDTA-SDS (TNx2) and guanidine hydrochloride (GHx2), or 100 percent ethanol. Rumen samples were collected via stomach intubation from 151 sheep at two time-points 2 weeks apart. Each sample was separated into four subsamples and preserved using the three preservation methods and the GRC method (n = 4x302). DNA was extracted and sequenced using Restriction Enzyme-Reduced Representation Sequencing to generate rumen microbial profiles. The profiles resulting from samples preserved using the solutions of TNx2, GHx2 and ethanol were examined for consistence against the profiles resulting from the GRC samples.