Extracellular DNA in environmental samples: Occurrence, extraction, quantification, and impact on microbial biodiversity assessment

2021 ◽  
Author(s):  
Sakcham Bairoliya ◽  
Jonas Koh Zhi Xiang ◽  
Bin Cao
Keyword(s):  
Dna Sequencing ◽  
Microbial Communities ◽  
Dna Extraction ◽  
Environmental Samples ◽  
Environmental Dna ◽  
Extracellular Dna ◽  
Biodiversity Assessment ◽  
Microbial Biodiversity ◽  
Technical Details ◽  
The Impact

Environmental DNA, i.e., DNA directly extracted from environmental samples, has been applied to understand microbial communities in the environments and to monitor contemporary biodiversity in the conservation context. Environmental DNA often contains both intracellular DNA (iDNA) and extracellular DNA (eDNA). eDNA can persist in the environment and complicate environmental DNA sequencing-based analyses of microbial communities and biodiversity. Although several studies acknowledged the impact of eDNA on DNA-based profiling of environmental communities, eDNA is still being neglected or ignored in most studies dealing with environmental samples. In this article, we summarize key findings on eDNA in environmental samples and discuss the methods used to extract and quantify eDNA as well as the importance of eDNA on the interpretation of experimental results. We then suggest several factors to consider when designing experiments and analyzing data to negate or determine the contribution of eDNA to environmental DNA-based community analyses. This field of research will be driven forward by: (i) carefully designing environmental DNA extraction pipelines by taking into consideration technical details in methods for eDNA extraction/removal and membrane-based filtration and concentration; (ii) quantifying eDNA in extracted environmental DNA using multiple methods including qPCR and fluorescent DNA binding dyes; (iii) carefully interpretating effect of eDNA on DNA-based community analyses at different taxonomic levels; and (iv) when possible, removing eDNA from environmental samples for DNA-based community analyses.

scholarly journals Profiling soil microbial communities with next-generation sequencing: the influence of DNA kit selection and technician technical expertise

2017 ◽  
Author(s):  
Taha Soliman ◽  
Sung-Yin Yang ◽  
Tomoko Yamazaki ◽  
Holger Jenke-Kodama
Keyword(s):  
Next Generation Sequencing ◽  
Microbial Communities ◽  
Dna Extraction ◽  
Dna Isolation ◽  
Soil Microbial Communities ◽  
Next Generation ◽  
Microbial Composition ◽  
Okinawa Island ◽  
The Impact ◽  
Generation Sequencing

Structure and diversity of microbial communities are an important research topic in biology, since microbes play essential roles in the ecology of various environments. Different DNA isolation protocols can lead to data bias and can affect results of next-generation sequencing. To evaluate the impact of protocols for DNA isolation from soil samples and also the influence of individual handling of samples, we compared results obtained by two researchers (R and T) using two different DNA extraction kits: (1) MO BIO PowerSoil® DNA Isolation kit (MO_R and MO_T) and (2) NucleoSpin® Soil kit (MN_R and MN_T). Samples were collected from six different sites on Okinawa Island, Japan. For all sites, differences in the results of microbial composition analyses (bacteria, archaea, fungi, and other eukaryotes), obtained by the two researchers using the two kits, were analyzed. For both researchers, the MN kit gave significantly higher yields of genomic DNA at all sites compared to the MO kit (ANOVA; P <0.006). In addition, operational taxonomic units for some phyla and classes were missed in some cases: Micrarchaea were detected only in the MN_T and MO_R analyses; the bacterial phylum Armatimonadetes was detected only in MO_R and MO_T; and WIM5 of the phylum Amoebozoa of eukaryotes was found only in the MO_T analysis. Our results suggest the possibility of handling bias; therefore, it is crucial that replicated DNA extraction be performed by at least two technicians for thorough microbial analyses and to obtain accurate estimates of microbial diversity.

scholarly journals Profiling soil microbial communities with next-generation sequencing: the influence of DNA kit selection and technician technical expertise

2017 ◽  
Author(s):  
Taha Soliman ◽  
Sung-Yin Yang ◽  
Tomoko Yamazaki ◽  
Holger Jenke-Kodama
Keyword(s):  
Next Generation Sequencing ◽  
Microbial Communities ◽  
Dna Extraction ◽  
Dna Isolation ◽  
Soil Microbial Communities ◽  
Next Generation ◽  
Microbial Composition ◽  
Okinawa Island ◽  
The Impact ◽  
Generation Sequencing

Structure and diversity of microbial communities are an important research topic in biology, since microbes play essential roles in the ecology of various environments. Different DNA isolation protocols can lead to data bias and can affect results of next-generation sequencing. To evaluate the impact of protocols for DNA isolation from soil samples and also the influence of individual handling of samples, we compared results obtained by two researchers (R and T) using two different DNA extraction kits: (1) MO BIO PowerSoil® DNA Isolation kit (MO_R and MO_T) and (2) NucleoSpin® Soil kit (MN_R and MN_T). Samples were collected from six different sites on Okinawa Island, Japan. For all sites, differences in the results of microbial composition analyses (bacteria, archaea, fungi, and other eukaryotes), obtained by the two researchers using the two kits, were analyzed. For both researchers, the MN kit gave significantly higher yields of genomic DNA at all sites compared to the MO kit (ANOVA; P <0.006). In addition, operational taxonomic units for some phyla and classes were missed in some cases: Micrarchaea were detected only in the MN_T and MO_R analyses; the bacterial phylum Armatimonadetes was detected only in MO_R and MO_T; and WIM5 of the phylum Amoebozoa of eukaryotes was found only in the MO_T analysis. Our results suggest the possibility of handling bias; therefore, it is crucial that replicated DNA extraction be performed by at least two technicians for thorough microbial analyses and to obtain accurate estimates of microbial diversity.

scholarly journals Profiling soil microbial communities with next-generation sequencing: the influence of DNA kit selection and technician technical expertise

PeerJ ◽  
2017 ◽  
Vol 5 ◽  
pp. e4178 ◽  
Author(s):  
Taha Soliman ◽  
Sung-Yin Yang ◽  
Tomoko Yamazaki ◽  
Holger Jenke-Kodama
Keyword(s):  
Next Generation Sequencing ◽  
Microbial Communities ◽  
Dna Extraction ◽  
Dna Isolation ◽  
Soil Microbial Communities ◽  
Next Generation ◽  
Microbial Composition ◽  
Okinawa Island ◽  
The Impact ◽  
Generation Sequencing

Structure and diversity of microbial communities are an important research topic in biology, since microbes play essential roles in the ecology of various environments. Different DNA isolation protocols can lead to data bias and can affect results of next-generation sequencing. To evaluate the impact of protocols for DNA isolation from soil samples and also the influence of individual handling of samples, we compared results obtained by two researchers (R and T) using two different DNA extraction kits: (1) MO BIO PowerSoil®DNA Isolation kit (MO_R and MO_T) and (2) NucleoSpin®Soil kit (MN_R and MN_T). Samples were collected from six different sites on Okinawa Island, Japan. For all sites, differences in the results of microbial composition analyses (bacteria, archaea, fungi, and other eukaryotes), obtained by the two researchers using the two kits, were analyzed. For both researchers, the MN kit gave significantly higher yields of genomic DNA at all sites compared to the MO kit (ANOVA;P < 0.006). In addition, operational taxonomic units for some phyla and classes were missed in some cases: Micrarchaea were detected only in the MN_T and MO_R analyses; the bacterial phylum Armatimonadetes was detected only in MO_R and MO_T; and WIM5 of the phylum Amoebozoa of eukaryotes was found only in the MO_T analysis. Our results suggest the possibility of handling bias; therefore, it is crucial that replicated DNA extraction be performed by at least two technicians for thorough microbial analyses and to obtain accurate estimates of microbial diversity.

scholarly journals Taxon appearance from extraction and amplification steps demonstrates the value of multiple controls in tick microbiota analysis

2019 ◽  
Author(s):  
Emilie Lejal ◽  
Agustín Estrada-Peña ◽  
Maud Marsot ◽  
Jean-François Cosson ◽  
Olivier Rué ◽  
...  
Keyword(s):  
Microbial Communities ◽  
Dna Extraction ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Dna Amplification ◽  
Community Diversity ◽  
Analytical Process ◽  
Sequencing Technologies ◽  
And Gender ◽  
The Impact

AbstractBackgroundThe development of high throughput sequencing technologies has substantially improved analysis of bacterial community diversity, composition, and functions. Over the last decade, high throughput sequencing has been used extensively to identify the diversity and composition of tick microbial communities. However, a growing number of studies are warning about the impact of contamination brought along the different steps of the analytical process, from DNA extraction to amplification. In low biomass samples, e.g. individual tick samples, these contaminants may represent a large part of the obtained sequences, and thus generate considerable errors in downstream analyses and in the interpretation of results. Most studies of tick microbiota either do not mention the inclusion of controls during the DNA extraction or amplification steps, or consider the lack of an electrophoresis signal as an absence of contamination. In this context, we aimed to assess the proportion of contaminant sequences resulting from these steps. We analyzed the microbiota of individual Ixodes ricinus ticks by including several categories of controls throughout the analytical process: crushing, DNA extraction, and DNA amplification.ResultsControls yielded a significant number of sequences (1,126 to 13,198 mean sequences, depending on the control category). Some operational taxonomic units (OTUs) detected in these controls belong to genera reported in previous tick microbiota studies. In this study, these OTUs accounted for 50.9% of the total number of sequences in our samples, and were considered contaminants. Contamination levels (i.e. the percentage of sequences belonging to OTUs identified as contaminants) varied with tick stage and gender: 76.3% of nymphs and 75% of males demonstrated contamination over 50%, while most females (65.7%) had rates lower than 20%. Contamination mainly corresponded to OTUs detected in crushing and DNA extraction controls, highlighting the importance of carefully controlling these steps.ConclusionHere, we showed that contaminant OTUs from extraction and amplification steps can represent more than half the total sequence yield in sequencing runs, and lead to unreliable results when characterizing tick microbial communities. We thus strongly advise the routine use of negative controls in tick microbiota studies, and more generally in studies involving low biomass samples.

scholarly journals Removal of Free Extracellular DNA from Environmental Samples by Ethidium Monoazide and Propidium Monoazide

2008 ◽  
Vol 74 (8) ◽  
pp. 2537-2539 ◽  
Author(s):  
Andreas O. Wagner ◽  
Cornelia Malin ◽  
Brigitte A. Knapp ◽  
Paul Illmer
Keyword(s):  
Dna Extraction ◽  
Environmental Samples ◽  
Extracellular Dna ◽  
Bacterial Cells ◽  
Propidium Monoazide ◽  
Extraction Techniques ◽  
Ethidium Monoazide ◽  
New Methods

ABSTRACT Recently, new DNA extraction techniques (using ethidium monoazide and propidium monoazide) have been developed to discriminate between alive and dead bacterial cells. Nevertheless, for complex environmental samples, no data are available yet. In the present study, these new methods were applied to anaerobic-fermentor sludge and the results were compared to a conventional microbiological approach.

scholarly journals Cyanobacterial Mats in Calcite-Precipitating Serpentinite-Hosted Alkaline Springs of the Voltri Massif, Italy

2020 ◽  
Vol 9 (1) ◽  
pp. 62
Author(s):  
Aysha Kamran ◽  
Kathrin Sauter ◽  
Andreas Reimer ◽  
Theresa Wacker ◽  
Joachim Reitner ◽  
...  
Keyword(s):  
Microbial Communities ◽  
Microbial Mats ◽  
Environmental Dna ◽  
Cyanobacterial Mats ◽  
Cyanobacterial Community ◽  
Mineral Precipitation ◽  
Community Based ◽  
Specific Adaptation ◽  
Carbonate Buildups ◽  
Generation Sequencing

(1) Background: Microbial communities in terrestrial, calcifying high-alkaline springs are not well understood. In this study, we investigate the structure and composition of microbial mats in ultrabasic (pH 10–12) serpentinite springs of the Voltri Massif (Italy). (2) Methods: Along with analysis of chemical and mineralogical parameters, environmental DNA was extracted and subjected to analysis of microbial communities based upon next-generation sequencing. (3) Results: Mineral precipitation and microbialite formation occurred, along with mat formation. Analysis of the serpentinite spring microbial community, based on Illumina sequencing of 16S rRNA amplicons, point to the relevance of alkaliphilic cyanobacteria, colonizing carbonate buildups. Cyanobacterial groups accounted for up to 45% of all retrieved sequences; 3–4 taxa were dominant, belonging to the filamentous groups of Leptolyngbyaceae, Oscillatoriales, and Pseudanabaenaceae. The cyanobacterial community found at these sites is clearly distinct from creek water sediment, highlighting their specific adaptation to these environments.

scholarly journals Amplicon-sequencing of raw milk microbiota: impact of DNA extraction and library-PCR

2021 ◽  
Author(s):  
Annemarie Siebert ◽  
Katharina Hofmann ◽  
Lena Staib ◽  
Etienne V. Doll ◽  
Siegfried Scherer ◽  
...  
Keyword(s):  
Sample Preparation ◽  
Dna Extraction ◽  
Raw Milk ◽  
Amplicon Sequencing ◽  
Rrna Gene ◽  
Bacterial Dna ◽  
Microbiome Analysis ◽  
Eukaryotic Dna ◽  
Selective Lysis ◽  
The Impact

Abstract The highly complex raw milk matrix challenges the sample preparation for amplicon-sequencing due to low bacterial counts and high amounts of eukaryotic DNA originating from the cow. In this study, we optimized the extraction of bacterial DNA from raw milk for microbiome analysis and evaluated the impact of cycle numbers in the library-PCR. The selective lysis of eukaryotic cells by proteinase K and digestion of released DNA before bacterial lysis resulted in a high reduction of mostly eukaryotic DNA and increased the proportion of bacterial DNA. Comparative microbiome analysis showed that a combined enzymatic and mechanical lysis procedure using the DNeasy® PowerFood® Microbial Kit with a modified protocol was best suitable to achieve high DNA quantities after library-PCR and broad coverage of detected bacterial biodiversity. Increasing cycle numbers during library-PCR systematically altered results for species and beta-diversity with a tendency to overrepresentation or underrepresentation of particular taxa. To limit PCR bias, high cycle numbers should thus be avoided. An optimized DNA extraction yielding sufficient bacterial DNA and enabling higher PCR efficiency is fundamental for successful library preparation. We suggest that a protocol using ethylenediaminetetraacetic acid (EDTA) to resolve casein micelles, selective lysis of somatic cells, extraction of bacterial DNA with a combination of mechanical and enzymatic lysis, and restriction of PCR cycles for analysis of raw milk microbiomes is optimal even for samples with low bacterial numbers. Key points • Sample preparation for high-throughput 16S rRNA gene sequencing of raw milk microbiota. • Reduction of eukaryotic DNA by enzymatic digestion. • Shift of detected microbiome caused by high cycle numbers in library-PCR.

scholarly journals A Low-Cost Assistive Robot for Children with Neurodevelopmental Disorders to Aid in Daily Living Activities

2021 ◽  
Vol 18 (8) ◽  
pp. 3974
Author(s):  
Roberto J. López-Sastre ◽  
Marcos Baptista-Ríos ◽  
Francisco Javier Acevedo-Rodríguez ◽  
Soraya Pacheco-da-Costa ◽  
Saturnino Maldonado-Bascón ◽  
...  
Keyword(s):  
Neurodevelopmental Disorders ◽  
Low Cost ◽  
Children And Youth ◽  
Proof Of Concept ◽  
Robotic Platform ◽  
Assistive Robot ◽  
Technical Details ◽  
The Impact ◽  
Near Future ◽  
Everyday Living

In this paper, we present a new low-cost robotic platform that has been explicitly developed to increase children with neurodevelopmental disorders’ involvement in the environment during everyday living activities. In order to support the children and youth with both the sequencing and learning of everyday living tasks, our robotic platform incorporates a sophisticated online action detection module that is capable of monitoring the acts performed by users. We explain all the technical details that allow many applications to be introduced to support individuals with functional diversity. We present this work as a proof of concept, which will enable an assessment of the impact that the developed technology may have on the collective of children and youth with neurodevelopmental disorders in the near future.

scholarly journals Benchmarking microbiome transformations favors experimental quantitative approaches to address compositionality and sampling depth biases

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Verónica Lloréns-Rico ◽  
Sara Vieira-Silva ◽  
Pedro J. Gonçalves ◽  
Gwen Falony ◽  
Jeroen Raes
Keyword(s):  
Microbial Communities ◽  
Quantitative Methods ◽  
Microbial Load ◽  
Metagenomic Sequencing ◽  
Sampling Depth ◽  
Microbiome Research ◽  
Microbiome Data ◽  
The Impact ◽  
Analytical Approaches ◽  
Quantitative Approaches

AbstractWhile metagenomic sequencing has become the tool of preference to study host-associated microbial communities, downstream analyses and clinical interpretation of microbiome data remains challenging due to the sparsity and compositionality of sequence matrices. Here, we evaluate both computational and experimental approaches proposed to mitigate the impact of these outstanding issues. Generating fecal metagenomes drawn from simulated microbial communities, we benchmark the performance of thirteen commonly used analytical approaches in terms of diversity estimation, identification of taxon-taxon associations, and assessment of taxon-metadata correlations under the challenge of varying microbial ecosystem loads. We find quantitative approaches including experimental procedures to incorporate microbial load variation in downstream analyses to perform significantly better than computational strategies designed to mitigate data compositionality and sparsity, not only improving the identification of true positive associations, but also reducing false positive detection. When analyzing simulated scenarios of low microbial load dysbiosis as observed in inflammatory pathologies, quantitative methods correcting for sampling depth show higher precision compared to uncorrected scaling. Overall, our findings advocate for a wider adoption of experimental quantitative approaches in microbiome research, yet also suggest preferred transformations for specific cases where determination of microbial load of samples is not feasible.

scholarly journals Microbial drivers of methane emissions from unrestored industrial salt ponds

2021 ◽  
Author(s):  
Jinglie Zhou ◽  
Susanna M. Theroux ◽  
Clifton P. Bueno de Mesquita ◽  
Wyatt H. Hartman ◽  
Ye Tian ◽  
...  
Keyword(s):  
Microbial Communities ◽  
Methane Flux ◽  
Methane Emissions ◽  
Metagenomic Data ◽  
Rrna Gene ◽  
Salt Ponds ◽  
Salt Pond ◽  
Reference Wetlands ◽  
Reference Wetland ◽  
The Impact

AbstractWetlands are important carbon (C) sinks, yet many have been destroyed and converted to other uses over the past few centuries, including industrial salt making. A renewed focus on wetland ecosystem services (e.g., flood control, and habitat) has resulted in numerous restoration efforts whose effect on microbial communities is largely unexplored. We investigated the impact of restoration on microbial community composition, metabolic functional potential, and methane flux by analyzing sediment cores from two unrestored former industrial salt ponds, a restored former industrial salt pond, and a reference wetland. We observed elevated methane emissions from unrestored salt ponds compared to the restored and reference wetlands, which was positively correlated with salinity and sulfate across all samples. 16S rRNA gene amplicon and shotgun metagenomic data revealed that the restored salt pond harbored communities more phylogenetically and functionally similar to the reference wetland than to unrestored ponds. Archaeal methanogenesis genes were positively correlated with methane flux, as were genes encoding enzymes for bacterial methylphosphonate degradation, suggesting methane is generated both from bacterial methylphosphonate degradation and archaeal methanogenesis in these sites. These observations demonstrate that restoration effectively converted industrial salt pond microbial communities back to compositions more similar to reference wetlands and lowered salinities, sulfate concentrations, and methane emissions.

Sign in / Sign up

Export Citation Format

Share Document