scholarly journals Performance evaluation of a new custom, multi-component DNA isolation method optimized for use in shotgun metagenomic sequencing-based aerosol microbiome research

2019 ◽  
Author(s):  
Kari Oline Bøifot ◽  
Jostein Gohli ◽  
Line Victoria Moen ◽  
Marius Dybwad
Keyword(s):  
Microbial Community ◽  
Real World ◽  
Dna Isolation ◽  
New Method ◽  
Metagenomic Sequencing ◽  
Isolation Method ◽  
Air Samples ◽  
Shotgun Metagenomic Sequencing ◽  
Isolation Methods ◽  
Microbiome Research

ABSTRACTBackgroundAerosol microbiome research advances our understanding of bioaerosols, including how airborne microorganisms affect our health and surrounding environment. Traditional microbiological/molecular methods are commonly used to study bioaerosols, but do not allow for generic, unbiased microbiome profiling. Recent studies have adopted shotgun metagenomic sequencing (SMS) to address this issue. However, SMS requires relatively large DNA inputs, which are challenging when studying low biomass air environments, and puts high requirements on air sampling, sample processing and DNA isolation protocols. Previous SMS studies have consequently adopted various mitigation strategies, including long-duration sampling, sample pooling, and whole genome amplification, each associated with some inherent drawbacks/limitations.ResultsHere, we demonstrate a new custom, multi-component DNA isolation method optimized for SMS-based aerosol microbiome research. The method achieves improved DNA yields from filter-collected air samples by isolating DNA from the entire filter extract, and ensures unbiased microbiome representation by combining chemical, enzymatic and mechanical lysis. Benchmarking against two state-of-the-art DNA isolation methods was performed with a mock microbial community and real-world subway air samples. All methods demonstrated similar performance regarding DNA yield and community representation with the mock community. However, with subway air samples, the new method obtained drastically improved DNA yields, while SMS revealed that the new method reported higher diversity and gave better taxonomic coverage. The new method involves intermediate filter extract separation into a pellet and supernatant fraction. Using subway air samples, we demonstrate that supernatant inclusion results in improved DNA yields. Furthermore, SMS of pellet and supernatant fractions revealed overall similar taxonomic composition but also identified differences that could bias the microbiome profile, emphasizing the importance of processing the entire filter extract.ConclusionsBy demonstrating and benchmarking a new DNA isolation method optimized for SMS-based aerosol microbiome research with both a mock microbial community and real-world air samples, this study contributes to improved selection, harmonization, and standardization of DNA isolation methods. Our findings highlight the importance of ensuring end-to-end sample integrity and using methods with well-defined performance characteristics. Taken together, the demonstrated performance characteristics suggest the new method could be used to improve the quality of SMS-based aerosol microbiome research in low biomass air environments.

scholarly journals Biased nicking of mitochondrial DNA during extraction can be avoided by choice of isolation method

2021 ◽  
Author(s):  
Bruno Marçal Repolês ◽  
Choco Michael Gorospe ◽  
Phong Tran ◽  
Anna Karin Nilsson ◽  
Paulina H. Wanrooij
Keyword(s):  
Skeletal Muscle ◽  
Mitochondrial Dna ◽  
Dna Isolation ◽  
Isolation Procedure ◽  
Isolation Method ◽  
Strand Breaks ◽  
Careful Choice ◽  
Isolation Methods ◽  
Solid Tissues ◽  
Long Range Pcr

AbstractThe integrity of mitochondrial DNA (mtDNA) isolated from solid tissues is critical for analyses such as long-range PCR. We show that a commonly-used DNA isolation procedure preferentially introduces strand breaks into the mtDNA extracted from the skeletal muscle of aged mice, while mtDNA from adult animals is less affected. We present a comparison of mtDNA isolation methods and identify one that avoids this biased loss of muscle mtDNA integrity. Our results highlight the importance of a careful choice of mtDNA isolation method and serve as a resource to researchers planning analysis of mtDNA isolated from solid tissues.

scholarly journals Shotgun metagenomic sequencing data of sunflower rhizosphere microbial community in South Africa

Data in Brief ◽  
2020 ◽  
Vol 31 ◽  
pp. 105831
Author(s):  
Olubukola Oluranti Babalola ◽  
Temitayo Tosin Alawiye ◽  
Carlos Rodriguez Lopez ◽  
Ayansina Segun Ayangbenro
Keyword(s):  
South Africa ◽  
Microbial Community ◽  
Metagenomic Sequencing ◽  
Sequencing Data ◽  
Shotgun Metagenomic Sequencing ◽  
Rhizosphere Microbial Community

scholarly journals Premature neonatal gut microbial community patterns supporting an epithelial TLR-mediated pathway for necrotizing enterocolitis

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Alexander G. Shaw ◽  
Kathleen Sim ◽  
Graham Rose ◽  
David J. Wooldridge ◽  
Ming-Shi Li ◽  
...  
Keyword(s):  
Microbial Community ◽  
Bayesian Regression ◽  
Validation Dataset ◽  
Metagenomic Sequencing ◽  
Faecal Microbiota ◽  
Community Patterns ◽  
Cpg Dna ◽  
Shotgun Metagenomic Sequencing ◽  
Low Levels ◽  
Stimulation Of

Abstract Background Necrotising enterocolitis (NEC) is a devastating bowel disease, primarily affecting premature infants, with a poorly understood aetiology. Prior studies have found associations in different cases with an overabundance of particular elements of the faecal microbiota (in particular Enterobacteriaceae or Clostridium perfringens), but there has been no explanation for the different results found in different cohorts. Immunological studies have indicated that stimulation of the TLR4 receptor is involved in development of NEC, with TLR4 signalling being antagonised by the activated TLR9 receptor. We speculated that differential stimulation of these two components of the signalling pathway by different microbiota might explain the dichotomous findings of microbiota-centered NEC studies. Here we used shotgun metagenomic sequencing and qPCR to characterise the faecal microbiota community of infants prior to NEC onset and in a set of matched controls. Bayesian regression was used to segregate cases from control samples using both microbial and clinical data. Results We found that the infants suffering from NEC fell into two groups based on their microbiota; one with low levels of CpG DNA in bacterial genomes and the other with high abundances of organisms expressing LPS. The identification of these characteristic communities was reproduced using an external metagenomic validation dataset. We propose that these two patterns represent the stimulation of a common pathway at extremes; the LPS-enriched microbiome suggesting overstimulation of TLR4, whilst a microbial community with low levels of CpG DNA suggests reduction of the counterbalance to TLR4 overstimulation. Conclusions The identified microbial community patterns support the concept of NEC resulting from TLR-mediated pathways. Identification of these signals suggests characteristics of the gastrointestinal microbial community to be avoided to prevent NEC. Potential pre- or pro-biotic treatments may be designed to optimise TLR signalling.

scholarly journals Metagenome-Assembled Genomes Isolated from a Human Fecal Diarrhea Sample

2021 ◽  
Vol 10 (19) ◽  
Author(s):  
Tshepiso Pleasure Ateba ◽  
Kazeem Adekunle Alayande ◽  
Ngoma Lubanza ◽  
Mulunda Mwanza
Keyword(s):  
Infectious Disease ◽  
Microbial Community ◽  
Fecal Sample ◽  
Metagenomic Sequencing ◽  
Affected Child ◽  
Shotgun Metagenomic Sequencing

ABSTRACT Diarrheal infection is the second leading infectious disease that is killing children under the age of 5 years. This study investigates the microbial community within a fecal sample from a diarrhea-affected child through shotgun metagenomic sequencing.

scholarly journals DEVELOPMENT OF DNA BIOSENSOR BASED ON SILVER NANOPARTICLES UV-Vis ABSORPTION SPECTRA FOR Escherichia coli DETECTION

2015 ◽  
Vol 2 (1) ◽  
pp. 382 ◽  
Author(s):  
Ruth Chrisnasari ◽  
Antonius Loren Wijaya ◽  
Maria Goretti Marianti Purwanto
Keyword(s):  
Escherichia Coli ◽  
Silver Nanoparticles ◽  
Absorption Spectra ◽  
Magnetic Beads ◽  
Dna Isolation ◽  
Dna Biosensor ◽  
Isolation Method ◽  
Isolation Methods ◽  
Spectra Properties ◽  
Magnetic Field Condition

<p>In this research we reported the synthesis of oligonucleotide-silver nanoparticle (OSN) conjugates and demonstrated their use along with magnetic beads as biosensor for Escherichia coli detection under magnetic field condition. Oligonucleotide DNA probes were conjugated on silver nanoparticles using alkanethiols linker. Two kinds of alkanethiols linker, 11-mercaptoundodecanoic acid (11-MUDA) and 16-mercaptophexadecanoic acid (16-MHDA) were compared to get the best probe conjugation yield and OSN UV-Vis absorption spectra properties. Three different methods of Escherichia coli DNA isolation i.e. Chen and Kuo (1993), Phenol Chloroform Isoamylalcohol (PCI) extraction and boiling lysis were also compared to explore the performance of the biosensor towards the DNA target purity. Detection process through hybridization between the DNA probe and the target was carried out at 55oC for 1 hour incubation time. The results showed that 16-MHDA gave higher conjugation yield and higher OSN UV-Vis absorption spectra than 11-MUDA. The biosensor was able to detect the presence of the DNA target which was isolated from the three isolation methods. The best detection signal was achieved by Chen and Kuo isolation method in which it could detect the presence of the DNA target up to 1.3 ng/µL.</p><p><br /><strong>Keywords</strong>: DNA biosensor, Silver Nanoparticles, Escherichia coli</p>

scholarly journals Metagenome Analysis of Surface Waters of the Shardara Reservoir, the Largest Artificial Reservoir in Southern Kazakhstan

2020 ◽  
Vol 9 (11) ◽  
Author(s):  
Madina S. Alexyuk ◽  
Andrey P. Bogoyavlenskiy ◽  
Pavel G. Alexyuk ◽  
Yergali S. Moldakhanov ◽  
Vladimir E. Berezin
Keyword(s):  
Microbial Community ◽  
Surface Waters ◽  
Metagenomic Analysis ◽  
Metagenomic Sequencing ◽  
Metagenome Analysis ◽  
Artificial Reservoir ◽  
Shotgun Metagenomic Sequencing ◽  
Hydropower Engineering

Here, we present a metagenomic analysis of the microflora of the surface waters of the Shardara reservoir, the largest artificial reservoir in Southern Kazakhstan, created to meet irrigation and hydropower engineering needs. In this case, shotgun metagenomic sequencing of the microbial community DNA was used.

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