Metagenomic DNA Extraction Research Articles

Gut metagenome in Insects and Its Potential in Agricultural and Industrial Applications

Metagenomics has greatly improved our understanding of microbial ecology by revealing the metagenomes of uncultured bacteria, including those associated with insects. These bacteria play important roles in insect defence, reproduction, and food absorption, which influences pest management tactics. Recent next generation sequencing advances in low-cost nucleotide and user-friendly bioinformatics have improved our ability to investigate microbial diversity in economically significant pests. Meta-omics technologies are now essential for defining microbial ecosystems, bio-surveillance, food safety, and commercial advances. Genome studies on insects and plants provide insights into pest control by discovering resistance genotypes and understanding stress-induced genomic changes. This review is focused on how herbivore specialists, such as the diamondback Plutella xylostella, adapt to host plants using gut microbiota. Metagenomic sequencing has discovered important bacteria-Enterobacter cloacae, Enterobacter asburiae, and Carnobacterium maltaromaticum- that help detoxify plant defences, breakdown cell walls, and synthesise amino acids. These findings indicate novel pest management tactics that target gut microbiota interactions. The paper continues with an overview of metagenomic DNA extraction, library creation and screening methods, especially on their applications in biotechnology and bioprocessing.

Melodious tuning of microbial dynamics in biofloc, cage, and pond culturing system: a study on Pangasius pangasius fish gut microbiome.

Aquaculture, a rapidly expanding sector, meets the global surging demand for aquatic food. Pangasius, a highly valued freshwater species, has seen a significant increase in demand due to its adaptability and potential for high yields, making it a promising candidate for aquaculture in India. This study investigates the gut microbiome composition of Pangasius pangasius fish cultured in three different systems (biofloc, cage, and pond). Metagenomic DNA extraction and 16S rRNA gene-targeted sequencing were performed. Outcomes revealed distinct microbial compositions across culture types, with significant differences in species richness and diversity, specifically in the biofloc system, compared to cages and ponds. Taxonomic analysis identified prevalent phyla such as Firmicutes and Fusobacteriota, with varying abundances among culture systems. The genus-level analysis highlighted dominant genera such as Cetobacterium and WWE3. Functional profiling indicated differences in enzymatic activity and metabolic pathways, emphasizing each culture sample type's unique microbial community structures. Notably, the microbiota from BF samples exhibited significant differences and unique metabolic pathways compared to the microbiota from C and P samples, which showed greater similarity and shared several common metabolic pathways. These findings highlight substantial differences in microbial diversity across the culturing systems, reflecting the microbiota's ability to adapt to specific environments and their potential role in promoting fish growth within those environments. Overall, this study provides insights into the gut microbiome diversity and functionality in Pangasius pangasius across different aquaculture environments, contributing to a better understanding of host-microbe interactions and aquaculture management strategies.

Impact of cell lysis treatment before saliva metagenomic DNA extraction on the oral microbiome and the associated resistome

Impact of cell lysis treatment before saliva metagenomic DNA extraction on the oral microbiome and the associated resistome

DNA metabarcoding technology for the identification of the fecal microbiome in patients with chronic stress

In the latest research, the concept of stress is associated with the deregulation of several biological systems sensitive to stress, such as the immune system, the microbiome, the endocrine system and neuroanatomical substrates. The objective of the research was to identify the fecal microbiome in patients diagnosed with chronic stress and in healthy patients through a metabarcoding analysis. The methodology used fecal samples collected from 20 patients with stress and 20 healthy patients. For the diagnosis of stress, psychological tools previously validated by external researchers were used. For metabarcoding analysis, metagenomic DNA extraction was performed from the fecal samples. Next Generation Illumina genetic sequencing targeting the 16S rDNA gene was then performed, followed by bioinformatic analysis using QUIME II software. The results, at the psychological test level, 20 people with chronic stress were diagnosed, on the other hand, at the metabarcoding level, specifically at the Gender level, the Asteroleplasma bacteria present only in the 20 healthy patients was molecularly identified. On the other hand, the bacteria Alistipes and Bifidobacterium were identified with greater predominance in the 20 patients with stress. Concluding, the bacteria Alistipes and Bifidobacterium are candidates as possible markers of the intestinal microbiome in patients with chronic stress, and the bacteria Asteroleplasma are candidates as a bacterial marker of the intestinal microbiome in healthy people. Finally, the identification of the microbiome in patients with stress opens a new path to understanding stress and its relationship to dysregulation with the microbiome.

Qualitative and quantitative assessment of metagenomic DNA isolated from diverse soil samples using different protocols

The quantity and quality of metagenomic DNA is an important consideration in search of novel microbial sources. For the metagenomic approach, different environmental samples have been used, though soil is the most preferred one. Several efforts have been made to isolate metagenomic DNA from different soil samples and accordingly, various protocols have been reported. Due to the variability in the soil samples, standardization of the protocol for the isolation of metagenomic DNA becomes a prerequisite. In the present study, an attempt has been made to compare a few selective protocols along with a commercial kit for the isolation of metagenomic DNA from soil samples representing nine different fruit orchards. The selected soil samples were from the orange, lemon, papaya, phalsa, banana, guava, litchi, custard apple, and starfruit growing region. The soil samples were characterized for their variability based on their texture, and nutrients. It was observed that protocol 3 with extraction buffer comprising chemicals like CTAB, SDS, CaCl2, PEG8000, and NaCl was found to be promising and reproducible irrespective of the different soil types. Further, to access the quality of the metagenomic DNA, PCR amplification with 16s rRNA primer and restriction analysis with Sau3A1 restriction endonuclease were performed. A linear correlation analysis between all four metagenomic DNA extraction methods and soil parameters was performed to see the effect of extraction methods on different soil types. The qualitative and quantitative aspect of the metagenomic DNA was also found to be dependent on the soil types.

Profiles of bacterial communities and environmental factors associated with proliferation of malaria vector mosquitoes within the Kenyan Coast.

Since Anopheles mosquitoes which transmit and maintain the malaria parasite breed in the outdoor environment, there is an urgent need to manage these mosquito breeding sites. In order to elaborate more on the ecological landscape of mosquito breeding sites, the bacterial community structure and their interactions with physicochemical factors in mosquito larval habitats was characterised in Kwale County (Kenya), where malaria is endemic. The physical characteristics and water physicochemical parameters of the habitats were determined and recorded. Water samples were also collected from the identified sites for total metagenomic DNA extraction in order to characterise the bacterial communities within the breeding sites. Sites where mosquito larvae were found were described as positive and those without mosquito larvae as negative. Electrical conductivity, total dissolved solids, salinity and ammonia were lower in the rainy season than in the dry season, which also coincided with a high proportion of positive sites. Pseudomonadota was the most common phyla recovered in all samples followed by Bacteroidota and then Actinomycetota. The presence or absence of mosquito larvae in a potential proliferation site was not related to the bacterial community structure in the sampled sites, but was positively correlated with bacterial richness and evenness. Generally, the presence of Anopheles mosquito larvae was found to be positively correlated with rainy season, bacterial richness and evenness, and negatively correlated with electrical conductivity, total dissolved solids, salinity and ammonia. The findings of this study have implications for predicting the potential of environmental water samples to become mosquito proliferation sites.

Identification of host-specific skin-mucus and gut microbiota in snakehead murrel (Channa striata) (Bloch, 1793) using metagenomics approach

Comparative analysis of metagenomic DNA extraction methods from gut microbiota of zebrafish (Danio rerio) for downstream next-generation sequencingVenetia D#;Rose, Tina Kollannoor Johny, Sarita Bhat

Comparison of Three DNA Extraction Kits for Assessment of Bacterial Diversity in Activated Sludge, Biofilm, and Anaerobic Digestate

Direct DNA analysis is the most widely used approach for microorganism characterization in natural and built environments; therefore, reliable and effective methods of nucleic acid extraction for samples from particular types of environments are needed. In this study, we compared three commercial kits for metagenomic DNA extraction from three types of biomass: activated sludge, biofilm, and anaerobic digestate. The yield, purity, and quality of DNA were measured, and the effect that the DNA kit had on the subsequent microbial community analysis was assessed with amplified ribosomal intergenic spacer analysis (ARISA). Amplicons were analyzed automatically utilizing capillary electrophoresis. For the activated sludge and digestate, the suggested kit is FastDNA™ Spin Kit for Soil (MP Biomedicals). This kit allowed the highest DNA yield to be obtained and provided the highest biodiversity. For biofilm with a high content of extracellular polymeric substances, the FavorPrep™ Soil DNA Isolation Mini Kit (FAVORGEN) is recommended. This kit allowed to obtain the highest biodiversity and provided the most reliable results of genetic distance assessment in this type of biomass.

Zederone Improves the Fecal Microbial Profile in Dementia Induced Rat Model: A First Report.

Dementia correlates with Alzheimer's disease, Parkinson's disease, frontotemporal and cerebrovascular diseases. There are supporting shreds of evidence on the pharmacological activity of curcuma caesia (Zingiberaceae family) for its antioxidant, antidepressant, analgesic, anticonvulsant, and anti-acetylcholinesterase effect. This study aims to analyze the fecal microbial profile in Zederone treated demented rat model. In our study, isolation and characterization of Zederone were carried out from curcuma caesia rhizomes, followed by estimation of its memory-enhancing effect on Aluminium-induced demented rat, which was evaluated by behavioural study on radial 8 arm maze. Moreover the detection of amyloid plaque formation was carried out using fluorescent microscopy of the congo red-stained rat brain tissues of the cerebral neocortex region. This study included eighteen female Wistar Albino rats that were divided into three groups that consisted of six rats in each group. The study of fecal microbial profile by metagenomic DNA extraction followed by next-generation sequencing was carried out to establish the correlation between gut microbes and amyloid plaques in dementia. Zederone could be characterized as pale yellow colored, needle-shaped crystals with 96.57% purity. This compound at 10 mg/kg body weight showed cognition improving capacity (p ≤ 0.0001) with a reduction of accumulated amyloid plaques that were detected in the demented group in fluorescence microscope and fecal microbiome study divulged an increased shift towards Lactobacillus genera in the treated group from Bacteroides in the demented group. This sesquiterpenoid compound would assist in the modulation of gut bacterial dysbiosis and act as a better therapeutic drug for dementia and other neurological disorders.

Improved method for the extraction of high-quality DNA from lignocellulosic compost samples for metagenomic studies.

The world economy is currently moving towards more sustainable approaches. Lignocellulosic biomass has been widely used as a substitute for fossil sources since it is considered a low-cost bio-renewable resource due to its abundance and continuous production. Compost habitats presenting high content of lignocellulosic biomass are considered a promising source of robust lignocellulose-degrading enzymes. Recently, several novel biocatalysts from different environments have been identified using metagenomic techniques. A key point of the metagenomics studies is the extraction and purification of nucleic acids. Nevertheless, the isolation of high molecular weight DNA from soil-like samples, such as compost, with the required quality for metagenomic approaches remains technically challenging, mainly due to the complex composition of the samples and the presence of contaminants like humic substances. In this work, a rapid and cost-effective protocol for metagenomic DNA extraction from compost samples composed of lignocellulosic residues and containing high content of humic substances was developed. The metagenomic DNA was considered as representative of the global environment and presented high quality (> 99% of humic acids effectively removed) and sufficient quantity (10.5-13.8µgg-1 of compost) for downstream applications, namely functional metagenomic studies. The protocol takes about 4h of bench work, and it can be performed using standard molecular biology equipment and reagents available in the laboratory. KEY POINTS/HIGHLIGHTS: • Metagenomic DNA was successfully extracted from compost samples rich in humic acids • The improved protocol was established by optimizing the cell lysis method and buffer • Complete removal of humic acids was achieved through the use of activated charcoal • The suitability of the DNA was proven by the construction of a metagenomic library.

Análisis comparativo de la diversidad microbiana usando un método efectivo y económico de extracción de mgDNA en un reactor batch con un sistema de transferencia directa de electrones entre especies

Recently, metagenomic DNA based analysis gained importance in fields such as environmental sciences and bioenergy, where mixed microbial communities embedded in complex matrixes, play a crucial role. Therefore, financially economical, and high quality metagenomic DNA extraction protocols are needed. In this work, a rapid and inexpensive method for high quality DNA extraction from a variety of complex samples was performed; the average DNA yield was 71.65 ng µL−1, with an average purity of 1.68 (A260/A280). The cost of extraction per sample was about 76 % less in comparison with commercial kits and the time needed to obtain the DNA pellets was about 4 hours. The DNA was suitable for 16S rRNA gene amplification by PCR and for next generation sequencing analysis, employing a MiSeq Ilumina platform. A high microbial diversity was detected in this study, and three main groups of bacteria were observed, which were developed according to the effect of the activated carbon had on them. The analysis performed showed a great difference between the samples, highlighting the differences between the microbial communities developed in the activated carbon biofilm and the bacteria detected in the reactor without activated carbon.

A novel approach to study cellulose digestion kinetics in biogas fermentation applying feed-stop method and artificial medium to investigate effects of saccharides

Anaerobic cellulose digestion kinetics was studied using a novel artificial medium, which allowed cellulose mono-fermentation with a complex biogas biocenosis. Semi-continuous cellulose mono-fermentation was successfully performed with this medium for more than 18 months achieving high organic loading rates of 8.3 gl−1d−1 and short hydraulic residence times of 9.6 days. Cellulose hydrolysis kinetics was studied by monitoring the substrate depletion after short feed-stops using the data obtained to establish a 1st order reaction model. This method was used to assess the cellulose hydrolysis kinetics under the influence of selected carbohydrates, of which a mixture of cellooligosaccharides showed the highest potential. A qPCR-Method was established for quantitative monitoring of selected cellulolytic clostridia based on the extraction of metagenomic DNA and comparison to standards from single cultures. The monitoring indicated consistent growth of these clostridia in the novel cultivation approach.

Quality of metagenomic DNA extracted for molecular identification of microorganisms from CSF samples of patients with suspected cerebrospinal meningitis in northern Nigeria

Background: Following an increase in the practice of starting antimicrobial therapy prior to clinical sample collection, the ability to confirm pathogenic microorganisms of bacterial meningitis has decreased by approximately 30%. Culture results may be false negative when fastidious or culture-resistant bacteria are involved or when patient samples are obtained after antimicrobial therapy has started. Molecular diagnosis using PCR can be performed directly on clinical samples after metagenomic DNA (mDNA) extraction not requiring live organisms for a positive result. The specific objectives of this study are to perform mDNA extraction directly from cerebrospinal fluids (CSF) using appropriate spin column method, and to determine the quality of the mDNA elute.Methodology: Cerebrospinal fluid specimens were collected from 210 patients with suspected acute cerebrospinal meningitis (CSM) in the Federal Capital Territory and some States in Northern Nigeria during the 2017 and 2018 outbreak seasons. Metagenomic DNA was extracted from approximately 200µL of CSF specimens using the Qiagen QIAamp(R) DNA Mini kit specific for bacterial agents only. DNA quality check was performed on all DNA elutes using fluorometric, spectrophotometric and agarose gel electrophoresis methods.Results: Of the 210 CSF samples analyzed microbiologically, Gram reaction was positive in 94 cases (44.8 %) but only 17 (8.1 %) were culture positive for two of the three major bacterial causes of meningitis. One hundred and eighty (85.7%) samples had DNA concentrations ≥ 0.005 ng/µL, 55 (30.6 %) of these had DNA purity (A260/A280) of ≥ 1.7, 103 (57.2%) had purity value between 1.0 - 1.69, 14 (7.8%) had value of 0.57 - 0.99, and 8 (4.4%) failed purity evaluation with value of 0.00 at A260/A280.Conclusion: The essence of mDNA extraction is multipurpose. A multiplex PCR can be performed on the extracted mDNA to interrogate the presence of microbial pathogens of interest using specific primers and probes (when applicable). Quality mDNA from CSF samples will ensure successful qPCR results for rapid and accurate detection of bacterial pathogens in meningitis. This will eliminate the challenges associated with traditional culture methods.
 Keywords: Meningitis, CSF, DNA Quality Check, Fluorometry

Contribution of sample processing to gut microbiome analysis in the model Lepidoptera, silkworm Bombyx mori

Microbes that live inside insects play various roles in host biology, ranging from nutrient supplementation to host defense. Although Lepidoptera (butterflies and moths) are one of the most diverse insect taxa and important in natural ecosystems, their microbiotas are little-studied, and to understand their structure and function, it is necessary to identify potential factors that affect microbiome analysis. Using a model organism, the silkworm Bombyx mori, we investigated the effects of different sample types (whole gut, gut content, gut tissue, starvation, or frass) and metagenomic DNA extraction methodologies (small-scale versus large-scale) on the composition and diversity of the caterpillar gut microbial communities. High-throughput 16S rRNA gene sequencing and computational analysis of the resulting data unraveled that DNA extraction has a large effect on the outcome of metagenomic analysis: significant biases were observed in estimates of community diversity and in the ratio between Gram-positive and Gram-negative bacteria. Furthermore, bacterial communities differed significantly among sample types. The gut content and whole gut samples differed least, both had a higher percentage of Enterococcus and Acinetobacter species; whereas the frass and starvation samples differed substantially from the whole gut and were poor representatives of the gut microbiome. Thus, we recommend a small-scale DNA extraction methodology for sampling the whole gut under normal insect rearing conditions whenever possible, as this approach provides the most accurate assessment of the gut microbiome. Our study highlights that evaluation of the optimal sample-processing approach should be the first step taken to confidently assess the contributions of microbiota to Lepidoptera.

Bacterial Operational Taxonomic Units Replace the Interactive Roles of Other Operational Taxonomic Units Under Strong Environmental Changes.

Background Microorganisms are an important component of an aquatic ecosystem and play a critical role in the biogeochemical cycle which influences the circulation of the materials and maintains the balance in aquatic ecosystems.Objective The seasonal variation along with the impact of anthropogenic activities, water quality, bacterial community composition and dynamics in the Loktak Lake, the largest freshwater lake of North East India, located in the Indo-Burma hotspot region was assessed during post-monsoon and winter season through metagenome analysis.Methods Five soil samples were collected during Post-monsoon and winter season from the Loktak Lake that had undergone different anthropogenic impacts. The metagenomic DNA of the soil samples was extracted using commercial metagenomic DNA extraction kits following the manufacturer’s instruction. The extracted DNA was used to prepare the NGS library and sequenced in the Illumina MiSeq platform.Results Metagenomics analysis reveals Proteobacteria as the predominant community followed by Acidobacteria and Actinobacteria. The presence of these groups of bacteria indicates nitrogen fixation, oxidation of iron, sulfur, methane, and source of novel antibiotic candidates. The bacterial members belonging to different groups were involved in various biogeochemical processes, including fixation of carbon and nitrogen, producing streptomycin, gramicidin and perform oxidation of sulfur, sulfide, ammonia, and methane.Conclusion The outcome of this study provides a valuable dataset representing a seasonal profile across various land use and analysis, targeting at establishing an understanding of how the microbial communities vary across the land use and the role of keystone taxa. The findings may contribute to searches for microbial bio-indicators as biodiversity markers for improving the aquatic ecosystem of the Loktak Lake.

Taxonomic and functional profiling of the microbial communities of Arabian Sea: A metagenomics approach

• Various methods for metagenomic DNA extraction from Seawater developed and optimized • First metagenomic study of the microbial communities of Arabian Sea water • Metagenomic sequencing revealed that majority of the microbes are uncultivable. • PICRUSt demonstrated potential xenobiotic degrading microbial communities in sea water.

Comparison of DNA Extraction Methods for Optimal Recovery of Metagenomic DNA from Human and Environmental Samples.

Metagenomics is the study of gene pool of an entire community in a particular niche. This provides valuable information about the functionality of host-microbe interaction in a biological ecosystem. Efficient metagenomic DNA extraction is a critical pre-requisite for a successful sequencing run in a metagenomic study. Although isolation of human stool metagenomic DNA is fairly standardized, the same protocol does not work as efficiently in fecal DNA from other organisms. In this study, we report a comparison of manual and commercial DNA extraction methods for diverse samples such as human stool, fish gut and soil. Fishes are known to have variable microbial diversity based on their food habits, so the study included two different varieties of fishes. A modified protocol for effective isolation of metagenomic DNA from human milk samples is also reported, highlighting critical precautions. Recent studies have emphasized the importance of studying functionality of human milk metagenome to understand its influence on infants' health. While manual method works well with most samples and therefore can be a method of choice for testing new samples, broad-range commercial kit offers advantage of high purity and quality. DNA extraction of different samples would go a long way in unraveling the unexplored association between microbes and host in a biological system.

Bacterial community structure in soils contaminated with electronic waste pollutants from Delhi NCR, India

Abstract Background Microbial community analysis of electronic waste (e-waste)-polluted environments is of interest to understand the effect of toxic e-waste pollutants on the soil microbial community and to evaluate novel microorganisms resisting the toxic environment. The present study aims to investigate the bacterial community structure in soils contaminated with e-waste from various sites of Loni and Mandoli (National Capital Region (NCR), India) where e-waste dumping and recycling activities are being carried out for many years. Results Interferences to soil metagenomic DNA extraction and PCR amplification were observed because of the presence of inhibiting components derived from circuit boards. Whole-metagenome sequencing on the Illumina MiSeq platform showed that the most abundant phyla were Proteobacteria and Firmicutes. Deltaproteobacteria and Betaproteobacteria were the most common classes under Proteobacteria. Denaturing gradient gel electrophoresis (DGGE) analysis of the bacterial 16S rRNA gene showed that e-waste contamination altered the soil bacterial composition and diversity. There was a decrease in the number of predominant bacterial groups like Proteobacteria and Firmicutes but emergence of Actinobacteria in the contaminated soil samples. Conclusions This is the first report describing the bacterial community structure of composite soil samples of e-waste-contaminated sites of Loni and Mandoli, Delhi NCR, India. The findings indicate that novel bacteria with potential bioremediating properties may be present in the e-waste-contaminated sites and hence need to be evaluated further. How to cite: Salam M, Varma A. Bacterial community structure in soils contaminated with electronic waste pollutants from Delhi NCR, India. Electron J Biotechnol 2019;41. https://doi.org/10.1016/j.ejbt.2019.07.003 .

DNA extraction and amplicon production strategies deeply inf\u200aluence the outcome of gut mycobiome studies

Microbial ecology studies are often performed through extraction of metagenomic DNA followed by amplification and sequencing of a marker. It is known that each step may bias the results. These biases have been explored for the study of bacterial communities, but rarely for fungi. Our aim was therefore to evaluate methods for the study of the gut mycobiome. We first evaluated DNA extraction methods in fungal cultures relevant to the gut. Afterwards, to assess how these methods would behave with an actual sample, stool from a donor was spiked with cells from the same cultures. We found that different extraction kits favour some species and bias against others. In terms of amplicon sequencing, we evaluated five primer sets, two for ITS2 and one for ITS1, 18S and 28S rRNA. Results showed that 18S rRNA outperformed the other markers: it was able to amplify all the species in the mock community and to discriminate among them. ITS primers showed both amplification and sequencing biases, the latter related to the variable length of the product. We identified several biases in the characterisation of the gut mycobiome and showed how crucial it is to be aware of these before drawing conclusions from the results of these studies.

Uncovering prokaryotic biodiversity within aerosols of the pristine Amazon forest

Biological aerosols (bioaerosol) are atmospheric particles that act as a dispersion unit of living organisms across the globe thereby affecting the biogeographic distribution of organisms. Despite their importance, there is virtually no knowledge about bioaerosols emitted by pristine forests. Here we provide the very first survey of the prokaryotic community of a bioaerosol collected inside pristine Amazon forest at 2 m above ground. Total atmospheric particles were collected at the Amazon Tall Tower Observatory, subjected to metagenomic DNA extraction and the prokaryotic diversity was determined by 16S rRNA gene amplicon sequencing. A total of 271,577 reads of 250 bp of the 16S rRNA gene amplicon were obtained. Only 27% of the reads could be classified using the 16S SILVA database. Most belonged to Proteobacteria, Actinobacteria and Firmicutes which is in good agreement with other bioaerosol studies. Further inspection of the reads using Blast searches and the 18S SILVA database revealed that most of the dataset was composed of Fungi sequences. The identified microbes suggest that the atmosphere may act as an important gateway to interchange bacteria between plants, soil and water ecosystems.