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Sample Processing Methods Impacts on Rumen Microbiome.

ABSTRACT: The standardization of collection and processing methods for rumen samples is crucial to reduce the level of errors that may affect the analysis and interpretation of the data. The aim of this study was to compare two processing methods and their impacts on the microbial community composition analysis, from material that was either immediately frozen or samples that were stored as cell pellets after removing the supernatant prior to freezing. Eight rumen-fistulated Brahman steers received chloroform as an antimethanogenic compound for 21 days. Rumen fluid samples (60 mL per animal) were collected using a probe covered with two layers of cheesecloth at 3 h post feeding at day 0 prior-treatment (control period) and day 21 of treatment. One sub-set of samples were placed in dry ice and stored at -80°C (Method 1) for subsequent DNA extraction, while a second subset of samples was centrifuged, the supernatant removed and the microbial pellet and rumen contents placed in dry ice and stored at -80°C (Method 2) prior to DNA extractions. Phylogenetic based methods (Illumina Miseq) targeting the 16S rRNA gene were used to characterize the bacterial and archaeal communities from both collection methods for the control and treatment periods. The results from this study showed that the chloroform treatment was significantly different for all beta diversity measures regardless of the processing method used. Significant differences in the relative abundances of some bacteria and archaea, such as Elusimicrobia, Fibrobacteres, Lentisphaerae, Spirochaetes, and Verrucomicrobia and Methanomassiliicoccaceae, were observed at higher levels in the Method 2. These microbial populations are known to have fragile cell wall structures and are susceptible to cell lysis. Regardless of the processing method used, both identified the key microbial groups and can be used to compare the relative shifts in the rumen microbiome between treatments. However, immediately freezing samples might alter the abundance of material from species that are more readily lysed and will not be suitable for studies that aim to assign absolute abundance values to these species within the rumen.

SUBMITTER: Martinez-Fernandez G

PROVIDER: S-EPMC6502991 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Sample Processing Methods Impacts on Rumen Microbiome.

Martinez-Fernandez Gonzalo G Denman Stuart E SE McSweeney Christopher S CS

Frontiers in microbiology 20190430

The standardization of collection and processing methods for rumen samples is crucial to reduce the level of errors that may affect the analysis and interpretation of the data. The aim of this study was to compare two processing methods and their impacts on the microbial community composition analysis, from material that was either immediately frozen or samples that were stored as cell pellets after removing the supernatant prior to freezing. Eight rumen-fistulated Brahman steers received chloro ...[more]

PMID: 31114550

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Project description:BackgroundThe 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.ResultsRumen 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.ConclusionsThe 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.

Dataset Information

Sample Processing Methods Impacts on Rumen Microbiome.

Publications

Sample Processing Methods Impacts on Rumen Microbiome.

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