Metagenomic DNA Extraction Research Articles

“Delineating bacterial community structure of rhizosphere metagenome from Kansal Forest”

Rhizosphere bacteria play crucial role in numerous biochemical and ecological processes ranging from biomass disintegration, mineralization, biological nitrogen fixation and de nitrification. The present investigation was carried out to delineate the microbial diversity of rhizosphere soil using metagenomic approach. Extraction of metagenomic DNA, PCR amplification and cloning of 16S rRNA genes resulted in library consisting of ~2000 clones. Sequencing of randomly selected 400 clones resulted in 352 unique sequences of 16S rRNA. Analysis of recovered sequences using BlastN (NCBI) revealed that ~47% of these sequences had uncultured representatives, uncultured Proteobacteria constituted ~8% of the total sequence diversity, uncultured acidobacteria comprised 5% of the total diversity among recovered clones. Other minor groups represent 2–5% of the total sequence diversity and include Bacillus and Pseudomonas . In addition, 1–2% of the total sequences also include uncultured Commensalibacter sp., Streptomyces sp., uncultured Gemmatimonadales , Halomonas (Gram-negative), uncultured Burkholderia sp., uncultured Pelomonas and other uncultured species. Phylogeny tree revealed that all sequence can be clustered into twelve main clusters indicating their diverse origin. Further clustering of these clones using QIIME resulted in 300 unique OTUs that constitutes 83.57% of the total sequence diversity, 52 clones were clustered as single OTU making a total 14.21%, while one sequence remain unclassified. At the Genus level, Bacillus, Pseudomonas and Staphylococcus demonstrated abundant presence in this soil. At species level Flavisolibacter tropicus of phylum Bacteriodetes showed maximum abundance followed by Bacillus cereus of phylum Firmicutes and Betaproteobacteria bacterium GR16-43 of phylum Proteobacteria.

Molecular Detection of the Predatory Bacterium Bdellovibrio bacteriovorus from Dental Biofilms

Introduction: The oral microbiome is a complex community whose composition results from multiple interactions among different microorganisms and with the host. Predatory prokaryotes are recognized as important balancing factors in different ecosystems. Among them Bdellovibrio bacteriovorus received special attention for its strong predatory behaviour against many human pathogens in the absence of any toxic or pathogenic effect, so that it was proposed as a live probiotic/antibiotic agent. This work aimed to evaluate if B. bacteriovorus is detected from samples of human oral and dental biofilm. Materials and Methods: Samples of oral and dental biofilm were obtained from 20 adults of both sexes and processed for extraction of metagenomic DNA, to be used as templates for B. bacteriovorus-specific PCR reactions. Specificity of amplification products was confirmed by sequencing. Results: All 20 dental biofilm samples and 12 of 20 (60%) oral biofilm samples were resulted PCR positive. The sequences of 17 of 32 PCR products (53.1%) showed 100% identity with the reference sequence; the sequences of 11 of 32 PCR products (34.4%) showed ≥99% identity, while the remaining 4 products (12.5%) showed identities ranging between

Comparative analysis of metagenomic DNA extraction methods from gut microbiota of zebrafish (Danio rerio) for downstream next-generation sequencing

Comparative analysis of metagenomic DNA extraction methods from gut microbiota of zebrafish (Danio rerio) for downstream next-generation sequencing

Effects of diet on the childhood gut microbiome and its implications for atopic dermatitis

Effects of diet on the childhood gut microbiome and its implications for atopic dermatitis

Progress of analytical tools and techniques for human gut microbiome research

Massive DNA sequencing studies have expanded our insights and understanding of the ecological and functional characteristics of the gut microbiome. Advanced sequencing technologies allow us to understand the close association of the gut microbiome with human health and critical illnesses. In the future, analyses of the gut microbiome will provide key information associating with human individual health, which will help provide personalized health care for diseases. Numerous molecular biological analysis tools have been rapidly developed and employed for the gut microbiome researches; however, methodological differences among researchers lead to inconsistent data, limiting extensive share of data. It is therefore very essential to standardize the current methodologies and establish appropriate pipelines for human gut microbiome research. Herein, we review the methods and procedures currently available for studying the human gut microbiome, including fecal sample collection, metagenomic DNA extraction, massive DNA sequencing, and data analyses with bioinformatics. We believe that this review will contribute to the progress of gut microbiome research in the clinical and practical aspects of human health.

Optimisation of protocol for effective detachment and selective recovery of the representative bacteria for extraction of metagenomic DNA from Eucalyptus spp. woodchips

For some environments such as planktonic/aqueous environments, the separation of bacteria cells from eukaryotic cells prior to DNA extraction using filtration is relatively straightforward. However, for woodchips, the bacteria are attached/embedded within the wood matrix, which prevents easy removal of bacterial cells. In this study, a method for the selective extraction of DNA from bacteria inhabiting Eucalyptus spp. woodchips has been developed. The objective was to compare milled and unmilled woodchips processed via three detachment methods, viz., sonication, vortexing and shaking followed by filtration using Teflon filters according to three relevant criteria: DNA yield, DNA purity and quality of DNA. Highest DNA yield was obtained by milling and vortexing for 10 min (77.50 ± 5.17 ng/μl), followed by milling and vortexing for 2 min (61.00 ± 6.56 ng/μl), unmilled and vortexing for 10 min (38.67 ± 5.17 ng/μl) and milled and shaking for 2 h (31.62 ± 5.17 ng/μl). The lowest DNA yield was obtained by using unmilled woodchips and 5 min of sonication treatment (7.00 ± 1.22 ng/μl). There was no significant difference in DNA purity for milled or unmilled woodchips processed via the three detachment methods. Duration of cell detachment treatment did not significantly influence DNA yield and purity. Following optimisation experiments, it was possible to extract bacterial DNA using milled woodchips and 10 minute vortexing devoid of DNA from the host background and other associated eukaryotes and of sufficient quality and quantity for metagenomic analysis.

Rapid and economical protocols for genomic and metagenomic DNA extraction from oak (Quercus brantii Lindl.)

Two new efficient, fast and low-cost metagenomic DNA extraction methods were developed for different Persian oak tissues (leaf, stem, root, and rhizospheric) and soil samples. The new “omics” studies on the genus Quercus are of importance to help finding efficient strategies for overcoming environmental challenges, and to do this, presence of efficient DNA extraction protocols for different Quercus species are very critical. The objective of the present study was to develop new efficient methods for extraction of metagenomic DNA (mDNA) from of Persian oak (Quercus brantii Lindl.) tissues. The efficiency of two newly developed mDNA extraction methods, including indirect SDS-based (ISB or concentrate method) and one spin column-based method (SCB) were compared to that of two classical direct methods, including CTAB-based and SDS-based methods, and two commercial mDNA extraction kits. The maximum average yield of mDNA for all samples (leaf, stem, root, bulk, and rhizospheric soils) was obtained by SCB (258 ng/μl) and ISB (189 ng/μl) methods, respectively. Successful PCR amplification for 16SrRNA and ITS sequence was consistently observed for ISB, SCB, and kit-extracted mDNAs, which confirmed the high purity of mDNA extracted by these methods. The new methods showed more than 96% quantitative PCR efficiency, and partial restriction digestion and metagenomic library construction confirmed the high efficiency of the newly developed methods. It could be concluded that two new protocols enhanced efficiency (yield, purity, and cost) of mDNA extraction from different tissues of Persian oak.

Metagenomic DNA Extraction of Natural Cellulose-Degrading Consortia

A natural cellulose-degrading consortium is able to efficiently convert various types of lignocellulose to organic products, which is considered a potential method for the production of biofuels and biochemicals from lignocellulose materials. Metagenomics is an efficient method for determining the complex microbiota structure and performing metabolic mechanism analysis. However, natural cellulose-degrading consortia not only have complicated microbiome compositions and metabolites but also have tight adsorption between cells and cellulose fibers, which could have a significant influence on metagenomic DNA extraction, leading to a reduction of the DNA concentration, integrity, and diversity. Here, we found that bacterial cells that bind to cellulose fibers were efficiently washed off by a high-salt solution treatment and that the wall-breaking ratios of the biological enzyme and mechanical homogenization method were significantly higher than those of grinding and the freeze-thaw method. These results helped to establish a new method for extraction of the consortia metagenome that efficiently decreased absorption between cells and cellulose fibers, reduced the adverse impact of metabolites on DNA extraction, and improved the quality of DNA extraction.

An improved method suitable for isolation of high-quality metagenomic DNA from diverse soils.

Standardization of metagenomic DNA extraction protocol is a pre-requisite for a successful metagenomic study aiming to screen and exploit the variety of microorganisms inhabiting a particular soil environment. Six methods reported earlier were used for isolation of metagenomic DNA in the present study. These methods suffered with regard to either poor yield or quality of DNA. Therefore, we developed an improved method for isolation of high-molecular weight and good quality metagenomic DNA from different soil samples. Our protocol combines the enzymatic (lysozyme and proteinase K) and chemical (CTAB and CaCl2) strategies to ensure efficient cell lysis and use of PEG and isopropanol for precipitation of humic impurities-free DNA. Our improved method gave high yield of good quality metagenomic DNA from diverse soils collected from garden, domestic waste dumping site, cellulose waste dumping site, sewage site, and tannery waste site. The good quality of the metagenomic DNA was evident by spectrophotometry data, PCR amplification of 16S rRNA gene and restriction digestion.

Rapid and efficient method to extract metagenomic DNA from estuarine sediments.

Metagenomic DNA from sediments of selective estuaries of Goa, India was extracted using a simple, fast, efficient and environment friendly method. The recovery of pure metagenomic DNA from our method was significantly high as compared to other well-known methods since the concentration of recovered metagenomic DNA ranged from 1185.1 to 4579.7µg/g of sediment. The purity of metagenomic DNA was also considerably high as the ratio of absorbance at 260 and 280nm ranged from 1.88 to 1.94. Therefore, the recovered metagenomic DNA was directly used to perform various molecular biology experiments viz. restriction digestion, PCR amplification, cloning and metagenomic library construction. This clearly proved that our protocol for metagenomic DNA extraction using silica gel efficiently removed the contaminants and prevented shearing of the metagenomic DNA. Thus, this modified method can be used to recover pure metagenomic DNA from various estuarine sediments in a rapid, efficient and eco-friendly manner.

Detection of Actinomyces spp. in cervical exudates from women with cervical intraepithelial neoplasia or cervical cancer.

Under certain circumstances, Actinomyces behaves as an opportunistic microorganism and can cause actinomycosis, a chronic and inflammatory granulomatous infection. The purpose of this project was to detect the presence of Actinomyces in cervical exudates from women with cervical intraepithelial neoplasia (CIN) and women with cervical cancer. Cervical samples from 92 women were divided into three groups: CIN, cervical cancer and healthy women. Metagenomic DNA extraction was performed following the Qiagen QIAamp Mini Kit protocol. A specific fragment (675 bp) was amplified by PCR in order to detect the presence of Actinomycetales. Samples in which Actinomycetales was detected were subjected to separate amplification reactions with primer pairs for A. israelii, A. viscosus, A. meyeri and A. odontolyticus. Amplified products were observed by 2 % agarose gel electrophoresis. Actinomyces were found in 10 % of women with CIN, 36.6 % of women with cervical cancer and 9 % of healthy women. The species identified in this study were A. meyeri in 14/92 samples (15.2 %), A. viscosus in 10/92 samples (10.8 %), A. odontolyticus in 4/92 samples (4.3 %) and A. israelii in 6/92 samples (6.5 %). Patients with cervical cancer had a higher prevalence of the presence of Actinomyces compared to the CIN and control groups. This is the first study in which a deliberate search of this genus has been performed in women with cervical pathologies. The use of specific primers for each species facilitated their detection in comparison with traditional isolation methods. More information is necessary to understand the molecular mechanisms involved in the complex role that bacterial communities may play in the development of cancer (and vice versa).

Comparison of Metagenomic DNA Extraction Methods for Soil Sediments of High Elevation Puga Hot Spring in Ladakh, India to Explore Bacterial Diversity

ABSTRACTExtraction of good-quality metagenomic DNA from extreme environments is quite challenging, particularly from high elevation hot spring sediments. Low microbial load, high humic acid content and other contaminants complicate the process of extraction of metagenomic DNA from hot spring sediments. In the present study, efficacy of five manual DNA extraction protocols with modifications has been evaluated for metagenomic DNA extraction from boron–sulfur rich high elevation Puga hot spring sediments. Best suited protocol was identified based on the cell lysis efficiency, DNA yield, humic acid content, PCR reproducibility and representation of bacterial diversity. Quantity as well as quality of crude metagenomic DNA differed remarkably between various protocols used and were not pure enough to give PCR amplification using 16S rRNA bacterial and archaeal primers. Crude metagenomic DNA extracted using five different DNA extraction protocols was purified using spin column based purification method. Even after purification, only three protocols C, D and E yielded metagenomic DNA that could be amplified using both archaeal and bacterial primers. To evaluate the degree of microbial diversity represented by protocols C, D and E, phylogenetic genes amplified were subjected to amplified ribosomal DNA restriction analysis (ARDRA) and denaturing gradient gel electrophoresis analysis (DGGE) analysis. ARDRA banding pattern of amplicons generated for all the three extraction protocols, i.e., C, D and E were found to be similar. DGGE of protocol E derived amplicons resulted in the similar number of dominant bands but a greater number of non-dominant bands, i.e., the highest microbial diversity in comparison to protocols C and D, respectively. In the present study, protocol E developed from Yeates et al. protocol has been found to be best in terms of DNA yield, DNA purity and bacterial diversity depiction associated with boron–sulfur rich sediment of high elevation hot springs.

Inexpensive metagenomic DNA extraction protocol with high quality from marine sediments contaminated by petroleum hydrocarbons.

Marine environments are a reservoir of relevant information on dangerous contaminants such as hydrocarbons, as well as microbial communities with probable degradation skills. However, to access microbial diversity, it is necessary to obtain high-quality DNA. An inexpensive, reliable, and effective metagenomic DNA (mgDNA) extraction protocol from marine sediments contaminated with petroleum hydrocarbons was established in this study from modifications to Zhou's protocol. The optimization included pretreatment of sediment with saline solutions for the removal of contaminants, a second precipitation and enzymatic degradation of RNA, followed by purification of mgDNA extracted by electroelution. The results obtained indicated that the modifications applied to 12 sediments with total petroleum hydrocarbon (TPH) concentrations from 22.6-174.3 (µg/g dry sediment) yielded 20.3-321.3 ng/µL mgDNA with A260/A280 and A260/A230 ratios of 1.75 ± 0.08 and 1.19 ± 0.22, respectively. The 16S rRNA amplification confirmed the purity of the mgDNA. The suitability of this mgDNA extraction protocol lies in the fact that all chemical solutions utilized are common in all molecular biology laboratories, and the use of dialysis membrane does not require any sophisticated or expensive equipment, only an electrophoretic chamber.

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

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 (>23kb) metagenomic DNA (260/280 ratio >1.8) with a good yield (55.8±3.8ng/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.

An Improved Procedure of the Metagenomic DNA Extraction from Saline Soil, Sediment and Salt

A new modified protocol has been developed for extracting pure community inhibitors-free DNA from saline soils, sediments and salts. Amplification of DNA from soil and sediment is often inhibited by copurified contaminants. A rapid, inexpensive, large-scale DNA extraction method involving minimal purification has been developed that is applicable to saline samples. Using a widely used a newly modified direct DNA extraction method proposed in this report, DNA was extracted from samples of Urmia Lake in diverse geological location in Iran and quantity of the DNA were examined. We developed an improved method to extract DNA include the combination of physical, chemical and mechanical lysis methods from saline samples. In the earlier reports, skim milk as an adsorption competitor was added to buffer DNA extract. In current study, we added skim milk to buffer DNA extraction. The results showed that skim milk was useful as an additive for extract DNA from saline samples. This method is applicable to molecular community analysis of saline samples which strongly adsorb DNA. The methods appear to have wide applicability in investigating molecular diversity and exploring functional genes from the total DNA. The extracted DNA was used to successfully amplify 16SrRNA region and functional genes. The amplicons were suitable for further applications such as diversity based analysis by denaturing gradient gel electrophoresis (DGGE) and cloning library.

An Improved Procedure of the Metagenomic DNA Extraction from Saline Soil, Sediment and Salt

A new modified protocol has been developed for extracting pure community inhibitors-free DNA from saline soils, sediments and salts. Amplification of DNA from soil and sediment is often inhibited by copurified contaminants. A rapid, inexpensive, large-scale DNA extraction method involving minimal purification has been developed that is applicable to saline samples. Using a widely used a newly modified direct DNA extraction method proposed in this report, DNA was extracted from samples of Urmia Lake in diverse geological location in Iran and quantity of the DNA were examined. We developed an improved method to extract DNA include the combination of physical, chemical and mechanical lysis methods from saline samples. In the earlier reports, skim milk as an adsorption competitor was added to buffer DNA extract. In current study, we added skim milk to buffer DNA extraction. The results showed that skim milk was useful as an additive for extract DNA from saline samples. This method is applicable to molecular community analysis of saline samples which strongly adsorb DNA. The methods appear to have wide applicability in investigating molecular diversity and exploring functional genes from the total DNA. The extracted DNA was used to successfully amplify 16SrRNA region and functional genes. The amplicons were suitable for further applications such as diversity based analysis by denaturing gradient gel electrophoresis (DGGE) and cloning library.

Response and resilience of soil microbial communities inhabiting in edible oil stress/contamination from industrial estates.

BackgroundGauging the microbial community structures and functions become imperative to understand the ecological processes. To understand the impact of long-term oil contamination on microbial community structure soil samples were taken from oil fields located in different industrial regions across Kadi, near Ahmedabad, India. Soil collected was hence used for metagenomic DNA extraction to study the capabilities of intrinsic microbial community in tolerating the oil perturbation.ResultsTaxonomic profiling was carried out by two different complementary approaches i.e. 16S rDNA and lowest common ancestor. The community profiling revealed the enrichment of phylum “Proteobacteria” and genus “Chromobacterium,” respectively for polluted soil sample. Our results indicated that soil microbial diversity (Shannon diversity index) decreased significantly with contamination. Further, assignment of obtained metagenome reads to Clusters of Orthologous Groups (COG) of protein and Kyoto Encyclopedia of Genes and Genomes (KEGG) hits revealed metabolic potential of indigenous microbial community. Enzymes were mapped on fatty acid biosynthesis pathway to elucidate their roles in possible catalytic reactions.ConclusionTo the best of our knowledge this is first study for influence of edible oil on soil microbial communities via shotgun sequencing. The results indicated that long-term oil contamination significantly affects soil microbial community structure by acting as an environmental filter to decrease the regional differences distinguishing soil microbial communities.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-016-0669-8) contains supplementary material, which is available to authorized users.

ALTERNATIF PENGGUNAAN SILIKA BENTONIT SEBAGAI PENGGANTI FENOL-KLOROFORM-ISOAMIL ALKOHOL DALAM EKSTRAKSI DNA SECARA LANGSUNG DARI TANAH HUTAN MANGROVE

Research on comparing the metagenomic DNA extraction from mangrove forest soil with phenol-chloroform-isoamyl alcohol and with silica bentonite was conducted. The purpose of this study was to find the differences in quality and quantity of metagenomic DNA extracted. The silica bentonite was compared to meet the Green Chemistry concept. The metagenomic DNA from these extractions were analyzed by agarose gel electrophoresis and UV-Vis spectrophotometry. The results of electrophoresis showed that metagenomic DNA extracted with silica bentonite were relatively higher in concentration than with phenol-chloroform-isoamyl alcohol; but integrity of their DNA were the same. The results of UV-Vis spectrophotometry analysis showed that A260/230 ratio of the phenol-chloroform-isoamyl alcohol extract had higher relative purity level to humic acid. The A260/280 ratio of the final elution of the silica bentonite extraction showed the highest relative purity level to protein.

<b>Bacterial diversity in bovine rumen by metagenomic 16S rDNA sequencing and scanning electron microscopy

The bacterial diversity by 16S rDNA partial sequencing and scanning electron microscope (SEM) of the rumen microbiome was characterized. Three Nellore bovines, cannulated at the rumen, were utilized. Liquid and solid fractions from the rumen content were processed for the extraction of metagenomic DNA and later 16S rDNA amplicons were utilized to construct the WGA library for further clone sequencing. Data were analyzed by MEGA and MOTUR (University of Michigan) softwares. Approximately 97.96% of operation taxonomic units (OTUs) were related to Bacteriodetes phylum and 2.04% of sequences were affiliated to Firmicutes phylum. In the case of Bacteriodetes, the great part of sequences (47.96%) was attributed to Prevotella genus. Bacteroidetes phylum was predominant in rumen content and the Prevotella genus was the most abundant, including diverse species related to this taxon. The bacterial morphological diversity associated to plant fibers was detected by SEM and showed its role in plant biomass deconstruction beyond the detection of microbiological interactions that involved protozoa

A Rapid and Economical Method for Efficient DNA Extraction from Diverse Soils Suitable for Metagenomic Applications.

A rapid, cost effective method of metagenomic DNA extraction from soil is a useful tool for environmental microbiology. The present work describes an improved method of DNA extraction namely “powdered glass method” from diverse soils. The method involves the use of sterile glass powder for cell lysis followed by addition of 1% powdered activated charcoal (PAC) as purifying agent to remove humic substances. The method yielded substantial DNA (5.87 ± 0.04 μg/g of soil) with high purity (A260/280: 1.76 ± 0.05) and reduced humic substances (A340: 0.047 ± 0.03). The quality of the extracted DNA was compared against five different methods based on 16S rDNA PCR amplification, BamHI digestion and validated using quantitative PCR. The digested DNA was used for a metagenomic library construction with the transformation efficiency of 4 X 106 CFU mL-1. Besides providing rapid, efficient and economical extraction of metgenomic DNA from diverse soils, this method’s applicability is also demonstrated for cultivated organisms (Gram positive B. subtilis NRRL-B-201, Gram negative E. coli MTCC40, and a microalgae C. sorokiniana UTEX#1666).