Dataset Information

Toward Best Practice in Livestock Microbiota Research: A Comprehensive Comparison of Sample Storage and DNA Extraction Strategies.

ABSTRACT: Understanding the roles of microorganisms in the animal gastrointestinal microenvironment is highly important for the development of effective strategies to manage and manipulate these microbial communities. In order to guide future animal gut microbiota research projects and standardization efforts, we have conducted a systematic comparison of 10 currently used sample preservation and DNA extraction approaches for pig and chicken microbiota samples and quantified their effects on bacterial DNA yield, quality, integrity, and on the resulting sequence-based bacterial composition estimates. The results showed how key stages of conducting a microbiota study, including the sample storage and DNA extraction, can substantially affect DNA recovery from the microbial community, and therefore, biological interpretation in a matrix-dependent manner. Our results highlight the fact that the influence of storage and extraction methods on the resulting microbial community structure differed by sample type, even within the same species. As the effects of these technical steps are potentially large compared with the real biological variability to be explained, standardization is crucial for accelerating progress in the area of livestock microbiota research. This study provided a framework to assist future animal gut microbiota research projects and standardization efforts.

SUBMITTER: Wegl G

PROVIDER: S-EPMC7940207 | biostudies-literature | 2021

REPOSITORIES: biostudies-literature

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Toward Best Practice in Livestock Microbiota Research: A Comprehensive Comparison of Sample Storage and DNA Extraction Strategies.

Wegl Gertrude G Grabner Nikolaus N Köstelbauer Andreas A Klose Viviana V Ghanbari Mahdi M

Frontiers in microbiology 20210223

Understanding the roles of microorganisms in the animal gastrointestinal microenvironment is highly important for the development of effective strategies to manage and manipulate these microbial communities. In order to guide future animal gut microbiota research projects and standardization efforts, we have conducted a systematic comparison of 10 currently used sample preservation and DNA extraction approaches for pig and chicken microbiota samples and quantified their effects on bacterial DNA ...[more]

PMID: 33708184

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Project description:BackgroundThere is growing interest in understanding gut microbiome dynamics, to increase the sustainability of livestock production systems and to better understand the dynamics that regulate antibiotic resistance genes (i.e., the resistome). High-throughput sequencing of RNA transcripts (RNA-seq) from microbial communities (metatranscriptome) allows an unprecedented opportunity to analyze the functional and taxonomical dynamics of the expressed microbiome and emerges as a highly informative approach. However, the isolation and preservation of high-quality RNA from livestock fecal samples remains highly challenging. This study aimed to determine the impact of the various sample storage and RNA extraction strategies on the recovery and integrity of microbial RNA extracted from selected livestock (chicken and pig) fecal samples.MethodsFecal samples from pigs and chicken were collected from conventional slaughterhouses. Two different storage buffers were used at two different storage temperatures. The extraction of total RNA was done using four different commercially available kits and RNA integrity/quality and concentration were measured using a Bioanalyzer 2100 system with RNA 6000 Nano kit (Agilent, Santa Clara, CA, USA). In addition, RT-qPCR was used to assess bacterial RNA quality and the level of host RNA contamination.ResultsThe quantity and quality of RNA differed by sample type (i.e., either pig or chicken) and most significantly by the extraction kit, with differences in the extraction method resulting in the least variability in pig feces samples and the most variability in chicken feces. Considering a tradeoff between the RNA yield and the RNA integrity and at the same time minimizing the amount of host RNA in the sample, a combination of storing the fecal samples in RNALater at either 4 °C (for 24 h) or -80 °C (up to 2 weeks) with extraction with PM kit (RNEasy Power Microbiome Kit) had the best performance for both chicken and pig samples.ConclusionOur findings provided a further emphasis on using a consistent methodology for sample storage, duration as well as a compatible RNA extraction approach. This is crucial as the impact of these technical steps can be potentially large compared with the real biological variability to be explained in microbiome and resistome studies.

Dataset Information

Toward Best Practice in Livestock Microbiota Research: A Comprehensive Comparison of Sample Storage and DNA Extraction Strategies.

Publications

Toward Best Practice in Livestock Microbiota Research: A Comprehensive Comparison of Sample Storage and DNA Extraction Strategies.

Similar Datasets

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