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

Abstract

Background: The use of rumen microbial community (RMC) profiles to predict methane emissions has driven interest in ruminal DNA preservation and extraction protocols that can be processed cheaply while also maintaining or improving DNA quality for RMC profiling. Our 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. This impedes researchers unable to access sufficient funding or infrastructure. 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% ethanol.

Results: 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 = 4 × 302). DNA was extracted and sequenced using Restriction Enzyme-Reduced Representation Sequencing to generate RMC profiles. Differences in DNA yield, quality and integrity, and sequencing metrics were observed across the methods (p < 0.0001). Ethanol exhibited poorer quality DNA (A260/A230 < 2) and more failed samples compared to the other methods. Samples preserved using the GRC method had smaller relative abundances in gram-negative genera Anaerovibrio, Bacteroides, Prevotella, Selenomonas, and Succiniclasticum, but larger relative abundances in the majority of 56 additional genera compared to TNx2 and GHx2. However, log₁₀ relative abundances across all genera and time-points for TNx2 and GHx2 were on average consistent (R² > 0.99) but slightly more variable compared to the GRC method. Relative abundances were moderately to highly correlated (0.68 ± 0.13) between methods for samples collected within a time-point, which was greater than the average correlation (0.17 ± 0.11) between time-points within a preservation method.

Conclusions: The two modified lysis buffers solutions (TNx2 and GHx2) proposed in this study were shown to be viable alternatives to the GRC method for RMC profiling in sheep. Use of these preservative solutions reduces cost and improves throughput associated with processing and sequencing ruminal samples. This development could significantly advance implementation of RMC profiles as a tool for breeding ruminant livestock.

Keywords: Genotyping-by-sequencing; PstI; RE-RRS; Rumen microbial profiles; Rumen microbiology; Superorganism.

Fig. 1

Schematic showing sampling, subsampling and processing of ruminal samples by the four preservation methods

Fig. 2

Electrophoresis gel of 12 sheep samples to show DNA integrity from four sample preservation methods. a GHx2 b EtOH c GRC d TNx2

Fig. 3

Relative abundance of bacterial and archaeal taxa in samples stored and extracted by the GRC, TNx2, GHx2 and EtOH methods. a bacteria with a gram-positive wall (black) and archaea (blue). b bacteria with a gram-negative wall (green). c Total sum of archaea, gram-positive and gram-negative taxa. Error bars represent ± one standard deviation of the raw means

Fig. 4

Comparison of means and standard deviations (SD) of log₁₀ relative abundances using three sample preservation methods. Diagonal graphs (top left to bottom right) represent standard deviation plotted against the mean for TNx2 (a), GHx2 (e) and the GRC method (i). Above the diagonal are the plots of means against each other for the three different preservations methods (b, c and f). Below the diagonal are the plots of standard deviations against each other for the three different preservations methods (d, g, h). Red lines represent the diagonal line of identity

Fig. 5

Matrix plot of first three PCs of log₁₀ relative abundances. Points are colored by preservative method in the upper panel (gold is GRC, blue is TNx2 and red is GHx2) and sampling round in the lower panel (round 1 is green and round 2 is purple)

Fig. 6

Pairwise correlation of log₁₀ relative abundances for each microbial taxa between different preservation methods within sampling rounds. Correlations for sampling round 1 are given in the first column (a,c,e) and correlations for sampling round 2 are given in the second column (b,d,f). Each label in the scatter plots gives the pairwise correlation estimate (between the two methods on the x-axis compared to the two methods marked on the y-axis) in log₁₀ relative abundances of each taxon for each sampling round. Taxon labels are colored based on whether the taxon is archaeal (blue), bacterial with a gram-positive wall (black) or bacterial with a gram-negative wall (green). The size of the taxon labels on the plots are proportional to the mean relative abundance of each taxon

Fig. 7

Correlation of log₁₀ relative abundance between sampling round 1 and 2 for samples preserved using different methods. a Correlation estimates between rounds for samples preserved using GRC compared to samples preserved using TNx2. b Correlation estimates between rounds for samples preserved using GRC compared to samples preserved using GHx2. Taxon labels are colored according to whether they are archaeal (blue), bacterial with a gram-positive wall (black) or bacterial with a gram-negative wall (green). The size of the taxon labels on the plots are proportional to the mean relative abundance of each taxon