Comparison of direct boiling method with commercial kits for extracting fecal microbiome DNA by Illumina sequencing of 16S rRNA tags

Peng, Xin; Yu, Ke-qiang; Deng, Guanhua; Jiang, Yunxia; Wang, Yu; Zhang, Guoxia; Zhou, Hongwei

doi:10.1016/j.mimet.2013.07.015

Cited by 87 publications

(64 citation statements)

References 17 publications

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“…Bacterial genomic DNA was extracted from mucosal samples using the DNA MAGNETICS and EXTRACT kit (Shenzhen BioEAsy Biotechnologies Co., Ltd., China), according to the manufacturer's instruction as previously validated (Peng et al, 2013). The barcoded V4F-GAGTGCCAGCMGCCGCGGTAA and V4R-GGACTACHVGGGTWTCTAAT primers were used to amplify bacterial 16S rRNA V4 fragments.…”

Section: Methodsmentioning

confidence: 99%

The Potential Effect of Oral Microbiota in the Prediction of Mucositis During Radiotherapy for Nasopharyngeal Carcinoma

et al. 2017

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BackgroundOral mucositis is probably the most debilitating complication that can arise in treating a patient with head and neck cancer. Little is known about the impacts of oral microbiota on the initiation and progression of mucositis.MethodsBased on 16S rRNA gene sequencing, dynamic changes in oral bacterial profile as well as correlations between the severity of mucositis and bacterial shifts during radiotherapy were investigated.FindingsOur results revealed that bacterial community structure altered progressively during radiation therapy, in parallel with a marked increase in the relative abundance of some Gram-negative bacteria. Patients who eventually developed severe mucositis harbored a significantly lower bacterial alpha diversity and higher abundance of Actinobacillus during the phase of erythema – patchy mucositis. Accordingly, a random forest model for predicting exacerbation of mucositis was generated, which achieved a high predictive accuracy (AUC) of 0.89.InterpretationOral microbiota changes correlate with the progression and aggravation of radiotherapy-induced mucositis in patients with nasopharyngeal carcinoma. Microbiota-based strategies can be used for the early prediction and prevention of the incidence of severe mucositis during radiotherapy.

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Section: Methodsmentioning

confidence: 99%

The Potential Effect of Oral Microbiota in the Prediction of Mucositis During Radiotherapy for Nasopharyngeal Carcinoma

et al. 2017

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“…[30] DNA concentrations were measured with a NanoDrop 2000 Bioanalyzer (Thermo Fisher Scientific, Inc., Waltham, MA). Samples were stored at −20 °C before qRT-PCR assays.…”

Section: Methodsmentioning

confidence: 99%

Increased Enterococcus faecalis infection is associated with clinically active Crohn disease

Zhou¹,

Chen²,

He³

et al. 2016

Medicine

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This study was performed to investigate the relationship between the abundance of pathogenic gut microbes in Chinese patients with inflammatory bowel disease (IBD) and disease severity.We collected clinical data and fecal samples from 47 therapy-naive Chinese patients with ulcerative colitis (UC), 67 patients with Crohn disease (CD), and 48 healthy volunteers. Bacteria levels of Fusobacterium species (spp), enterotoxigenic Bacteroides fragilis (B fragilis), enteropathogenic Escherichia coli (E coli), and Enterococcus faecalis (E faecalis) were assessed by quantitative real-time PCR (qRT-PCR). Spearman correlation coefficients were calculated to test associations between bacterial content and clinical parameters.Compared to healthy controls, the levels of both Fusobacterium spp and E faecalis were significantly increased in the feces of patients with IBD (P < 0.01). B fragilis levels were higher (P < 0.05) and E faecalis levels lower (P < 0.05) in patients with CD compared to those with UC. Increased E faecalis colonization in CD associated positively with disease activity (P = 0.015), Crohn disease activity index (CDAI; R = 0.3118, P = 0.0108), and fecal calprotectin (P = 0.016).E faecalis and Fusobacterium spp are significantly enriched in patients with IBD, and increased E faecalis infection is associated with clinically active CD.

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“…The variations in the resultant microbial profiles in different studies due to methodological disparities make comparisons difficult and not very meaningful (Mai and Draganov, 2009). Although the results of a few studies available to date have shown no significant effects of different DNA extraction methods on the resultant bacterial profiles of biological samples (Peng et al, 2013, Wagner Mackenzie et al, 2015, a number of workers have recognised and called for a consensus and a universal DNA extraction method for microbiome analyses to overcome the challenges and misrepresentation inherent in using disparate techniques (Abusleme et al, 2014, Claassen et al, 2013, Henderson et al, 2013, Kennedy et al, 2014, Lazarevic et al, 2013, Maukonen et al, 2012, Walker et al, 2015, Yuan et al, 2012.…”

Section: Accepted M Manuscriptmentioning

confidence: 99%

The yield and quality of cellular and bacterial DNA extracts from human oral rinse samples are variably affected by the cell lysis methodology

Sohrabi

Nair

Samaranayake

et al. 2016

Journal of Microbiological Methods

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Q., The yield and quality of cellular and bacterial DNA extracts from human oral rinse samples are variably affected by the cell lysis methodology, Journal of Microbiological Methods (2016Methods ( ), doi: 10.1016Methods ( /j.mimet.2016 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. A C C E P T E DM A N U S C R I P T ACCEPTED MANUSCRIPT A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT Highlights Lysozyme-achromopeptidase cell lysis yielded the highest gDNA level  Lysozyme-zirconium beads cell lysis extracted the highest level of bacterial DNA  Lysozyme-zirconium beads cell lysis yielded the highest level of Firmicutes A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT ABSTRACTRecent culture-independent studies have enabled detailed mapping of human microbiome that has not been hitherto achievable by culture-based methods. DNA extraction is a key element of bacterial culture-independent studies that critically impacts on the outcome of the detected microbial profile. Despite the variations in DNA extraction methods described in the literature, no standardized technique is available for the purpose of microbiome profiling.Hence, standardization of DNA extraction methods is urgently needed to yield comparable data from different studies. We examined the effect of eight different cell lysis protocols on the yield and quality of the extracted DNA from oral rinse samples. These samples were exposed to cell lysis techniques based on enzymatic, mechanical, and a combination of enzymatic-mechanical methods. The outcome measures evaluated were total bacterial population, Firmicutes levels and human DNA contamination (in terms of surrogate GAPDH levels). We noted that all three parameters were significantly affected by the method of cell lysis employed. Although the highest yield of gDNA was obtained using lysozymeachromopeptidase method, the lysozyme-zirconium beads method yielded the peak quantity of total bacterial DNA and Firmicutes with a lower degree of GAPDH contamination compared with the other methods. Taken together our data clearly points to an urgent need for a consensus, standardized DNA extraction technique to evaluate the oral microbiome using oral rinse samples. Further, if Firmicutes levels are the focus of investigation in oral rinse microbiome analyses then the lysozyme-zirconium bead method would be the method of choice in preference to others.

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Comparison of direct boiling method with commercial kits for extracting fecal microbiome DNA by Illumina sequencing of 16S rRNA tags

Cited by 87 publications

References 17 publications

The Potential Effect of Oral Microbiota in the Prediction of Mucositis During Radiotherapy for Nasopharyngeal Carcinoma

The Potential Effect of Oral Microbiota in the Prediction of Mucositis During Radiotherapy for Nasopharyngeal Carcinoma

Increased Enterococcus faecalis infection is associated with clinically active Crohn disease

The yield and quality of cellular and bacterial DNA extracts from human oral rinse samples are variably affected by the cell lysis methodology

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