Improved extraction of PCR-quality community DNA from digesta and fecal samples

Yu, Zhongtang; Morrison, Mark

doi:10.2144/04365st04

Cited by 1,370 publications

(1,053 citation statements)

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“…DNA was extracted using MoBio Power Soil DNA kits. In order to maximize the yield and purity of manure DNA extracts, the repeated bead beading plus column extraction method (RBBC) was used (Yu and Morrison, 2004). The RBBC method combines bead beating with a lysis buffer containing sodium dodecyl sulfate and EDTA.…”

Section: Dna Extractionmentioning

confidence: 99%

Fate and transport of tylosin-resistant bacteria and macrolide resistance genes in artificially drained agricultural fields receiving swine manure

Luby

Moorman

Soupir

2016

Science of The Total Environment

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Application of manure from swine treated with antibiotics introduces antibiotics and antibiotic resistance genes to soil with the potential for further movement in drainage water, which may contribute to the increase in antibiotic resistance in non-agricultural settings. We compared losses of antibiotic-resistant Enterococcus and macrolide-resistance (erm and msrA) genes in water draining from plots with or without swine manure application under chisel plow and no till conditions. Concentrations of ermB,ermC and ermF were all > 10 9 copies g − 1 in manure from tylosin-treated swine, and application of this manure resulted in short-term increases in the abundance of these genes in soil. Abundances of ermB, ermC and ermF in manured soil returned to levels identified in non-manured control plots by the spring following manure application. Tillage practices yielded no significant differences (p > 0.10) in enterococci or erm gene concentrations in drainage water and were therefore combined for further analysis. While enterococci and tylosin-resistant enterococci concentrations in drainage water showed no effects of manure application, ermB and ermF concentrations in drainage water from manured plots were significantly higher (p < 0.01) than concentrations coming from non-manured plots. ErmB and ermF were detected in 78% and 44%, respectively, of water samples draining from plots receiving manure. Although ermC had the highest concentrations of the three genes in drainage water, there was no effect of manure application on ermC abundance. MsrA was not detected in manure, soil or water. This study is the first to report significant increases in abundance of resistance genes in waters draining from agricultural land due to manure application. H I G H L I G H T S• Higher levels of ermB and ermF in drainage are attributable to manure.• Fall manure application resulted in transient increases in soil enterococci.• Manure application did not affect enterococci levels in drainage water. Application of manure from swine treated with antibiotics introduces antibiotics and antibiotic resistance genes to soil with the potential for further movement in drainage water, which may contribute to the increase in antibiotic resistance in non-agricultural settings. We compared losses of antibiotic-resistant Enterococcus and macrolide-resistance (erm and msrA) genes in water draining from plots with or without swine manure application under chisel plow and no till conditions. Concentrations of ermB, ermC and ermF were all N10 9 copies g −1 in manure from tylosin-treated swine, and application of this manure resulted in short-term increases in the abundance of these genes in soil. Abundances of ermB, ermC and ermF in manured soil returned to levels identified in non-manured control plots by the spring following manure application. Tillage practices yielded no significant differences (p N 0.10) in enterococci or erm gene concentrations in drainage water and were therefore combined for further analysis. While enterococci and tylosin...

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Section: Dna Extractionmentioning

confidence: 99%

Fate and transport of tylosin-resistant bacteria and macrolide resistance genes in artificially drained agricultural fields receiving swine manure

Luby

Moorman

Soupir

2016

Science of The Total Environment

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“…To evaluate the relative performance of our current method, we compared it with other commercially available metagenomic DNA isolation kits for human feces [11], [16], [21]. As instructed by kit manufacturers, various defined amount of fecal sample were used for commercial methods A, B, C, and D. Notably, low yields of DNA were obtained using these methods in comparison to that using current method (Table 1).…”

Section: Resultsmentioning

confidence: 99%

“…Removal of fibers and undigested particles from feces is difficult, which in turn affects overall quality and quantity of metagenomic DNA being isolated [14], [15], [16]. Moreover, the presence of these impurities also compromises efficient lysis of microbial cells.…”

Section: Introductionmentioning

confidence: 99%

“…Additionally, human genomic DNA remnants in metagenomic DNA affect the metagenomic sequence data output, thereby increasing per base sequencing cost [6]. To overcome these challenges, many metagenomic DNA extraction protocols are being standardized, which include phenol/chloroform enzymatic lysis and freeze thaw [11], [16], [17], [18], [19], [20], [21], [22]. These kits have improved the quality of metagenomic DNA.…”

Section: Introductionmentioning

confidence: 99%

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An Improved Methodology to Overcome Key Issues in Human Fecal Metagenomic DNA Extraction

Kumar

Gupta

et al. 2016

Genomics, Proteomics &Amp; Bioinformatics

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Microbes are ubiquitously distributed in nature, and recent culture-independent studies have highlighted the significance of gut microbiota in human health and disease. Fecal DNA is the primary source for the majority of human gut microbiome studies. However, further improvement is needed to obtain fecal metagenomic DNA with sufficient amount and good quality but low host genomic DNA contamination. In the current study, we demonstrate a quick, robust, unbiased, and cost-effective method for the isolation of high molecular weight (>23 kb) metagenomic DNA (260/280 ratio >1.8) with a good yield (55.8 ± 3.8 ng/mg of feces). We also confirm that there is very low human genomic DNA contamination (eubacterial: human genomic DNA marker genes = 227.9:1) in the human feces. The newly-developed method robustly performs for fresh as well as stored fecal samples as demonstrated by 16S rRNA gene sequencing using 454 FLX+. Moreover, 16S rRNA gene analysis indicated that compared to other DNA extraction methods tested, the fecal metagenomic DNA isolated with current methodology retains species richness and does not show microbial diversity biases, which is further confirmed by qPCR with a known quantity of spike-in genomes. Overall, our data highlight a protocol with a balance between quality, amount, user-friendliness, and cost effectiveness for its suitability toward usage for culture-independent analysis of the human gut microbiome, which provides a robust solution to overcome key issues associated with fecal metagenomic DNA isolation in human gut microbiome studies.

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“…Sampling, metagenomic library preparation and sequencing DNA was extracted from rumen fluid samples using the repeat bead beating and column purification method of Yu and Morrison (2004). DNA was fragmented to an average size of 300 bp using a Bioruptor (Diagenode, Seraing, Belgium).…”

Section: Methodsmentioning

confidence: 99%

Erratum: Divergent functional isoforms drive niche specialisation for nutrient acquisition and use in rumen microbiome

Rubino¹,

Carberry²,

Waters³

et al. 2017

The ISME Journal

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Many microbes in complex competitive environments share genes for acquiring and utilising nutrients, questioning whether niche specialisation exists and if so, how it is maintained. We investigated the genomic signatures of niche specialisation in the rumen microbiome, a highly competitive, anaerobic environment, with limited nutrient availability determined by the biomass consumed by the host. We generated individual metagenomic libraries from 14 cows fed an ad libitum diet of grass silage and calculated functional isoform diversity for each microbial gene identified. The animal replicates were used to calculate confidence intervals to test for differences in diversity of functional isoforms between microbes that may drive niche specialisation. We identified 153 genes with significant differences in functional isoform diversity between the two most abundant bacterial genera in the rumen (Prevotella and Clostridium). We found Prevotella possesses a more diverse range of isoforms capable of degrading hemicellulose, whereas Clostridium for cellulose. Furthermore, significant differences were observed in key metabolic processes indicating that isoform diversity plays an important role in maintaining their niche specialisation. The methods presented represent a novel approach for untangling complex interactions between microorganisms in natural environments and have resulted in an expanded catalogue of gene targets central to rumen cellulosic biomass degradation.

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Improved extraction of PCR-quality community DNA from digesta and fecal samples

Cited by 1,370 publications

References 20 publications

Fate and transport of tylosin-resistant bacteria and macrolide resistance genes in artificially drained agricultural fields receiving swine manure

Fate and transport of tylosin-resistant bacteria and macrolide resistance genes in artificially drained agricultural fields receiving swine manure

An Improved Methodology to Overcome Key Issues in Human Fecal Metagenomic DNA Extraction

Erratum: Divergent functional isoforms drive niche specialisation for nutrient acquisition and use in rumen microbiome

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