Tendentious effects of automated and manual metagenomic DNA purification protocols on broiler gut microbiome taxonomic profiling

Fidler, Gabor; Tolnai, Emese; Stágel, Anikó; Remenyik, Judit; Stündl, László; Gál, Ferenc; Bíró, Sándor; Paholcsek, Melinda

doi:10.1038/s41598-020-60304-y

Cited by 24 publications

(29 citation statements)

References 60 publications

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“…The extraction of high-quality DNA from complex matrices is often a challenging task, and this, in turn, may affect its accurate quantification [25]. Apart the extraction efficiency, another critical issue is the integrity of the extracted DNA [42], which is usually measured by the DIN using the Agilent 2200 TapeStation system [43].…”

Section: Resultsmentioning

confidence: 99%

Accurate quantification of bacterial abundance in metagenomic DNAs accounting for variable DNA integrity levels

et al. 2020

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The quantification of the total microbial content in metagenomic samples is critical for investigating the interplay between the microbiome and its host, as well as for assessing the accuracy and precision of the relative microbial composition which can be strongly biased in low microbial biomass samples. In the present study, we demonstrate that digital droplet PCR (ddPCR) can provide accurate quantification of the total copy number of the 16S rRNA gene, the gene usually exploited for assessing total bacterial abundance in metagenomic DNA samples. Notably, using DNA templates with different integrity levels, as measured by the DNA integrity number (DIN), we demonstrated that 16S rRNA copy number quantification is strongly affected by DNA quality and determined a precise correlation between quantification underestimation and DNA degradation levels. Therefore, we propose an input DNA mass correction, according to the observed DIN value, which could prevent inaccurate quantification of 16S copy number in degraded metagenomic DNAs. Our results highlight that a preliminary evaluation of the metagenomic DNA integrity should be considered before performing metagenomic analyses of different samples, both for the assessment of the reliability of observed differential abundances in different conditions and to obtain significant functional insights.

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Section: Resultsmentioning

confidence: 99%

Accurate quantification of bacterial abundance in metagenomic DNAs accounting for variable DNA integrity levels

et al. 2020

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“…Therefore, we cannot claim that the microbiota profiles observed here represent those from a larger number of individuals. However, we pooled our samples to ensure that an adequate sample volume was available for multiple comparisons, an approach that has been used for prior gut microbiota studies in human and animal gut microbiota research ( Lu et al, 2015 ; Knudsen et al, 2016 ; Fidler et al, 2020 ).…”

Section: Discussionmentioning

confidence: 99%

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

Wegl

Grabner²,

Köstelbauer³

et al. 2021

Front. Microbiol.

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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.

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“…Bacterial cell suspensions (BS) were prepared from 7 g of each broiler stool sample. Then, 7 ml of sterile PBS buffer (Thermo Fisher Scientific, MD, USA) was added to each of the samples, and they were homogenized for 4 min (by vortexing at 350 rpm) (126). The samples were centrifuged for 5 min at 500 Â g. Supernatants were collected, and the washing step was repeated 2 times.…”

Section: Methodsmentioning

confidence: 99%

Nutraceuticals Induced Changes in the Broiler Gastrointestinal Tract Microbiota

et al. 2021

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Effects of nutraceuticals on the intestinal microbiota are receiving increased attention; however, there are few studies investigating their effects on broiler meat production. The aim of this study was to implement feeding strategies and carry out a comprehensive trial examining the interplay between natural biologically active compounds such as carotenoids, anthocyanins, fermentable oligosaccharides, and synbiotics and the gastrointestinal tract microbiota. Our feeding program was applied to an intensive production system with a flock of 1,080 Ross 308 broilers. Aging induced significant changes through the feeding experiment. Nutraceuticals were shown to modulate broiler intestinal diversity and differentially enriched Lactobacillus, Enterococcus, Campylobacter, and Streptococcus in the core microbiome during the different stages of broiler rearing. Additionally, they did not remarkably affect animal growth performance; nevertheless, a positive correlation was found between body weight and Corynebacteriales and Pseudomonadales. Furthermore, a diet high in carotenoid, fermentable oligosaccharide, and anthocyanin contents affected the number of beneficial genera such as Faecalibacterium, Lactobacillus, Blautia, and Ruminococcus. With this comprehensive trial, we revealed that nutraceuticals induced modulations in broiler gastrointestinal tract microbiota. We believe that plant-derived immunostimulants, recycled from plant food waste products, can supplement antibiotic-free broiler meat production. IMPORTANCE In this trial, nutraceuticals were manufactured from waste products of food industry processing of Hungarian red sweet pepper and sour cherry and incorporated into the diet of poultry to investigate their effects on broilers’ growth and the broiler gastrointestinal tract microbiota. To avoid the generation of food waste products, we believe that this approach can be developed into a sustainable, green approach that can be implemented in commercial antibiotic-free poultry to provide safe and high-quality meat.

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Tendentious effects of automated and manual metagenomic DNA purification protocols on broiler gut microbiome taxonomic profiling

Cited by 24 publications

References 60 publications

Accurate quantification of bacterial abundance in metagenomic DNAs accounting for variable DNA integrity levels

Accurate quantification of bacterial abundance in metagenomic DNAs accounting for variable DNA integrity levels

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

Nutraceuticals Induced Changes in the Broiler Gastrointestinal Tract Microbiota

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