Pre-centrifugation before DNA extraction mitigates extraction efficiency reduction of environmental DNA caused by the preservative solution (benzalkonium chloride) remaining in the filters

Limnology ◽  
2021 ◽  
Author(s):  
Satsuki Tsuji ◽  
Ryohei Nakao ◽  
Minoru Saito ◽  
Toshifumi Minamoto ◽  
Yoshihisa Akamatsu
Keyword(s):  
Dna Extraction ◽  
Benzalkonium Chloride ◽  
Extraction Efficiency ◽  
Environmental Dna ◽  
Preservative Solution

scholarly journals Influence of DNA extraction kits on the fungal DNA metabarcoding for freshwater environmental DNA

2021 ◽  
Author(s):  
Shunsuke Matsuoka ◽  
Yoriko Sugiyama ◽  
Mariko Nagano ◽  
Hideyuki Doi
Keyword(s):  
Dna Extraction ◽  
Environmental Dna ◽  
Operational Taxonomic Unit ◽  
Pcr Inhibition ◽  
Dna Metabarcoding ◽  
Fungal Dna ◽  
Diversity Studies ◽  
Microbial Dna ◽  
Methodological Studies ◽  
Biodiversity Analysis

Background: Environmental DNA (eDNA) metabarcoding is a rapidly expanding technique for efficient biodiversity monitoring, especially of animals. Recently, the usefulness of aquatic eDNA in monitoring the diversity of both terrestrial and aquatic fungi has been suggested. In eDNA studies, different experimental factors, such as DNA extraction kits or methods, can affect the subsequent analyses and the results of DNA metabarcoding. However, few methodological studies have been carried out on eDNA of fungi, and little is known about how experimental procedures can affect the results of biodiversity analysis. In this study, we focused on the effect of the DNA extraction method on fungal DNA metabarcoding using freshwater samples obtained from rivers and lakes. Methods: DNA was extracted from freshwater samples using the DNeasy PowerSoil kit, which is mainly used to extract microbial DNA from soil, and the DNeasy Blood & Tissue kit, which is commonly used for eDNA studies on animals. We then compared PCR inhibition and fungal DNA metabarcoding results [i.e., operational taxonomic unit (OTU) number and composition] of the extracted samples. Results: No PCR inhibition was detected in any of the samples, and no significant differences in the number of OTUs and OTU compositions were detected between the samples processed using different kits. These results indicate that both DNA extraction kits may provide similar diversity results for the river and lake samples evaluated in this study. Therefore, it may be possible to evaluate the diversity of fungi using a unified experimental method, even with samples obtained for diversity studies on other taxa such as those of animals.

scholarly journals Environmental DNA monitoring of noble crayfish Astacus astacus: Comparison and refining of methodology

2021 ◽  
Vol 4 ◽  
Author(s):  
David Strand ◽  
Stein Johnsen ◽  
Frode Fossøy ◽  
Johannes Rusch ◽  
Brett Sandercock ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Brown Trout ◽  
Detection Probability ◽  
Environmental Dna ◽  
Northern Pike ◽  
Freshwater Crayfish ◽  
Astacus Astacus ◽  
European Perch ◽  
Detection Frequency ◽  
Noble Crayfish

During the past decade, environmental DNA (eDNA) methodology has become an important non-invasive tool to monitor aquatic micro- and macro-organisms, including freshwater crayfish. In Europe, noble crayfish Astacus astacus is the most widespread native freshwater crayfish. However, the species is threatened in its entire distribution range. It is therefore included on the International Union for Conservation Nature (IUCN) red list, and on several national red lists. Reliable monitoring is essential for implementation of conservation measures. For crayfish, traditional population trends have been obtained from catch per unit effort (CPUE) data. In order to successfully apply and use eDNA monitoring for noble crayfish, or any species, it is a prerequisite to know the strengths and weaknesses of the applied methods and how they perform compared to traditional methodology. Sampling strategy and analysis methodology also depends on choice of species to be monitored, and which questions to be answered. Further, refinement of the employed methods may improve the detection probability for eDNA monitoring. Here we report the results from 1) a recently published study on noble crayfish eDNA monitoring (Johnsen et al. 2020) and 2) an ongoing study comparing and optimising the methods used for monitoring noble crayfish. 1) We compared eDNA monitoring (transects with ten 5L samples) with traditional trapping (transects with 50 traps) for noble crayfish in lentic habitats, in order to evaluate detection probability and if eDNA concentration correlates with relative density of crayfish. We also compared two commonly used analytical methods [quantitative real-time PCR (qPCR) and droplet digital PCR (ddPCR)] for eDNA monitoring. We found that qPCR outperformed ddPCR in detection frequency (Fig. 1), most likely due to some inhibition in the ddPCR analysis. eDNA monitoring provided reliable presence/absence data for noble crayfish, even in lakes with very low crayfish densities. Detection frequency increased with increasing CPUE (Fig. 1). However, we did not observe any correlation between relative crayfish densities and eDNA concentrations of crayfish. eDNA concentrations were consistently very low, even in lakes with very high crayfish densities. For lakes with very low crayfish densities, we estimated that ~5 samples (5L samples) are needed for 95 % detection likelihood, while for lakes with high densities 2 samples were needed. 2) We compared two eDNA sampling strategies (sampling from bottom or the surface), commonly used for crayfish or fish in Norway to investigate how both strategies perform. The sampled filters were divided and two DNA extraction protocols were evaluated (CTAB based vs Column based). We found that the DNA yield was higher from the column based DNA extraction protocol, and that eDNA concentrations from fish (brown trout Salmon trutta, northern pike Esox lucius and European perch Perca fluviatilis) were significantly higher than for crayfish. For crayfish and brown trout, there was little difference between detection probability for bottom and surface samples, while for northern pike and European perch the detection probability was higher for the bottom samples. Currently, we are analysing eDNA samples collected with glass fibre filters and NatureMetrix filters for noble crayfish in both lentic and lotic habitats and the preliminary results will be presented. We conclude that eDNA monitoring cannot substitute CPUE monitoring for freshwater crayfish, but it offers reliable presence-absence data, provided sufficient sampling efforts. Thus, it is suitable for large scale monitoring of threatened crayfish and combined with eDNA analysis of alien crayfish and diseases such as crayfish plague, this is a cost-efficient supplement offering a more holistic approach for aquatic environments and native crayfish conservation. Furthermore, the synergy effect of using collected eDNA samples from different projects to monitor additional species is substantial.

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.

DNA extraction efficiency from soil as affected by pyrolysis temperature and extractable organic carbon of high-ash biochar

2017 ◽  
Vol 115 ◽  
pp. 129-136 ◽  
Author(s):  
Zhongmin Dai ◽  
Tara M. Webster ◽  
Akio Enders ◽  
Kelly L. Hanley ◽  
Jianming Xu ◽  
...  
Keyword(s):  
Organic Carbon ◽  
Dna Extraction ◽  
Extraction Efficiency ◽  
Pyrolysis Temperature

scholarly journals Comparing sediment DNA extraction methods for assessing organic enrichment associated with marine aquaculture

PeerJ ◽  
2020 ◽  
Vol 8 ◽  
pp. e10231
Author(s):  
John K. Pearman ◽  
Nigel B. Keeley ◽  
Susanna A. Wood ◽  
Olivier Laroche ◽  
Anastasija Zaiko ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Community Composition ◽  
Oncorhynchus Tshawytscha ◽  
Environmental Dna ◽  
Illumina Miseq ◽  
Ecological Impacts ◽  
Biotic Index ◽  
Soil Dna ◽  
Additional Advantage ◽  
Marine Aquaculture

Marine sediments contain a high diversity of micro- and macro-organisms which are important in the functioning of biogeochemical cycles. Traditionally, anthropogenic perturbation has been investigated by identifying macro-organism responses along gradients. Environmental DNA (eDNA) analyses have recently been advocated as a rapid and cost-effective approach to measuring ecological impacts and efforts are underway to incorporate eDNA tools into monitoring. Before these methods can replace or complement existing methods, robustness and repeatability of each analytical step has to be demonstrated. One area that requires further investigation is the selection of sediment DNA extraction method. Environmental DNA sediment samples were obtained along a disturbance gradient adjacent to a Chinook (Oncorhynchus tshawytscha) salmon farm in Otanerau Bay, New Zealand. DNA was extracted using four extraction kits (Qiagen DNeasy PowerSoil, Qiagen DNeasy PowerSoil Pro, Qiagen RNeasy PowerSoil Total RNA/DNA extraction/elution and Favorgen FavorPrep Soil DNA Isolation Midi Kit) and three sediment volumes (0.25, 2, and 5 g). Prokaryotic and eukaryotic communities were amplified using primers targeting the 16S and 18S ribosomal RNA genes, respectively, and were sequenced on an Illumina MiSeq. Diversity and community composition estimates were obtained from each extraction kit, as well as their relative performance in established metabarcoding biotic indices. Differences were observed in the quality and quantity of the extracted DNA amongst kits with the two Qiagen DNeasy PowerSoil kits performing best. Significant differences were observed in both prokaryotes and eukaryotes (p < 0.001) richness among kits. A small proportion of amplicon sequence variants (ASVs) were shared amongst the kits (~3%) although these shared ASVs accounted for the majority of sequence reads (prokaryotes: 59.9%, eukaryotes: 67.2%). Differences were observed in the richness and relative abundance of taxonomic classes revealed with each kit. Multivariate analysis showed that there was a significant interaction between “distance” from the farm and “kit” in explaining the composition of the communities, with the distance from the farm being a stronger determinant of community composition. Comparison of the kits against the bacterial and eukaryotic metabarcoding biotic index suggested that all kits showed similar patterns along the environmental gradient. Overall, we advocate for the use of Qiagen DNeasy PowerSoil kits for use when characterizing prokaryotic and eukaryotic eDNA from marine farm sediments. We base this conclusion on the higher DNA quality values and richness achieved with these kits compared to the other kits/amounts investigated in this study. The additional advantage of the PowerSoil Kits is that DNA extractions can be performed using an extractor robot, offering additional standardization and reproducibility of results.

scholarly journals Suppression of environmental DNA degradation in water samples associated with different storage temperature and period using benzalkonium chloride

2020 ◽  
Vol 18 (8) ◽  
pp. 437-445 ◽  
Author(s):  
Teruhiko Takahara ◽  
Junya Taguchi ◽  
Satoshi Yamagishi ◽  
Hideyuki Doi ◽  
Shigeki Ogata ◽  
...  
Keyword(s):  
Water Samples ◽  
Benzalkonium Chloride ◽  
Storage Temperature ◽  
Environmental Dna ◽  
Dna Degradation

scholarly journals The room temperature preservation of filtered environmental DNA samples and assimilation into a phenol–chloroform–isoamyl alcohol DNA extraction

2014 ◽  
Vol 15 (1) ◽  
pp. 168-176 ◽  
Author(s):  
Mark A. Renshaw ◽  
Brett P. Olds ◽  
Christopher L. Jerde ◽  
Margaret M. McVeigh ◽  
David M. Lodge
Keyword(s):  
Dna Extraction ◽  
Room Temperature ◽  
Environmental Dna ◽  
Isoamyl Alcohol

scholarly journals Optimizing an eDNA protocol for monitoring endangered Chinook Salmon in the San Francisco Estuary: balancing sensitivity, cost and time

2019 ◽  
Author(s):  
Thiago M. Sanches ◽  
Andrea M. Schreier
Keyword(s):  
Dna Extraction ◽  
San Francisco ◽  
Chinook Salmon ◽  
Magnetic Beads ◽  
Oncorhynchus Tshawytscha ◽  
Environmental Dna ◽  
San Francisco Estuary ◽  
Time Cost ◽  
Dna Yield ◽  
Pcr Inhibitor

AbstractEnvironmental DNA (eDNA) analysis has gained traction as a precise and cost effective method for species and waterways management. To date, publications on eDNA protocol optimization have focused primarily on DNA yield. Therefore, it has not been possible to evaluate the cost and speed of specific components of the eDNA protocol, such as water filtration and DNA extraction method when designing or choosing an eDNA pipeline. At the same time, these two parameters are essential for the experimental design of a project. Here we evaluate and rank different eDNA protocols in the context of Chinook salmon (Oncorhynchus tshawytscha) eDNA detection in an aquatic environment, the San Francisco Estuary. We present a comprehensive evaluation of multiple eDNA protocol parameters, balancing time, cost and DNA yield. For estuarine waters, which are challenging for eDNA studies due to high turbidity, variable salinity, and the presence of PCR inhibitors, we find that a protocol combining glass filters and magnetic beads, along with an extra step for PCR inhibitor removal, is the method that best balances time, cost, and yield. In addition, we provide a generalized decision tree for determining the optimal eDNA protocol for other studies on aquatic systems. Our findings should be applicable to most aquatic environments and provide a clear guide for determining which eDNA pipeline should be used for a given environmental condition.Author SummaryThe use of environmental DNA (eDNA) analysis for monitoring wildlife has steadily grown in recent years. Though, due to differences in the ecology of the environment studied and the novelty of the technique, eDNA currently shows a lack of standards compared to other fields. Here we take a deep look into each step of an eDNA assay, looking at common protocols and comparing their efficiencies in terms of time to process the samples, cost and how much DNA is recovered. We then analyze the data to provide a concise interpretation of best practices given different project constraints. For the conditions of the San Francisco Estuary we suggest the use of glass fiber filtration, the use of paramagnetic beads for DNA extraction and the use of a secondary inhibitor removal. We expect our findings to provide better support for managers to decide their standards ahead of project submission not only for estuarine conditions but for other waterine conditions alike.

scholarly journals Filtration extraction method using microfluidic channel for measuring environmental DNA

2021 ◽  
Author(s):  
Takashi Fukuzawa ◽  
Yuichi Kameda ◽  
Hisao Nagata ◽  
Naofumi Nishizawa ◽  
Hideyuki Doi
Keyword(s):  
Water Sample ◽  
River Water ◽  
Dna Extraction ◽  
Water Samples ◽  
Extraction Method ◽  
Microfluidic Channel ◽  
Pcr Amplification ◽  
Environmental Dna ◽  
Laboratory Analysis ◽  
River Water Samples

The environmental DNA (eDNA) method, which is widely applied for biomonitoring, is limited to laboratory analysis and processing. In this study, we developed a filtration/extraction component using a microfluidic channel, Biryu-Chip (BC), and a filtration/extraction method, BC method, to minimize the volume of the sample necessary for DNA extraction and subsequent PCR amplification. We tested the performance of the BC method and compared it with the Sterivex filtration/extraction method using aquarium and river water samples. We observed that using the BC method, the same concentration of the extracted DNA was obtained with 1/20–1/40 of the filtration volume of the Sterivex method, suggesting that the BC method can be widely used for eDNA measurement. In addition, we could perform on-site measurements of eDNA within 30 min using a mobile PCR device. Using the BC method, filtration and extraction could be performed easily and quickly. The PCR results obtained by the BC method were similar to those obtained by the Sterivex method. The BC method required fewer steps and therefore, the risk of DNA contamination could be reduced. When combined with a mobile PCR, the BC method can be applied to easily detect eDNA within 30 min from a few 10 mL of the water sample, even on-site.

scholarly journals Freeze-Thaw Pretreatment Can Improve Efficiency of Bacterial DNA Extraction From Meconium

2021 ◽  
Vol 12 ◽  
Author(s):  
Yuntian Xin ◽  
Jingxian Xie ◽  
Bingru Nan ◽  
Chen Tang ◽  
Yunshan Xiao ◽  
...  
Keyword(s):  
Species Diversity ◽  
Liquid Nitrogen ◽  
Dna Extraction ◽  
Extraction Efficiency ◽  
Bacterial Colonization ◽  
Care Hospital ◽  
Bacterial Dna ◽  
Freeze Thaw ◽  
Low Efficiency ◽  
Bacterial Dna Extraction

Although the presence of live microbes in utero remains under debate, newborn gastrointestinal bacteria are undoubtedly important to infant health. Measuring bacteria in meconium is an ideal strategy to understand this issue; however, the low efficiency of bacterial DNA extraction from meconium has limited its utilization. This study aims to improve the efficiency of bacterial DNA extraction from meconium, which generally has low levels of microflora but high levels of PCR inhibitors in the viscous matrix. The research was approved by the ethical committee of the Xiamen Maternity and Child Health Care Hospital, Xiamen, China. All the mothers delivered naturally, and their newborns were healthy. Meconium samples passed by the newborns within 24 h were collected. Each sample was scraped off of a sterile diaper, transferred to a 5-ml sterile tube, and stored at −80°C. For the assay, a freeze-thawing sample preparation protocol was designed, in which a meconium-InhibitEX buffer mixture was intentionally frozen 1–3 times at −20°C, −80°C, and (or) in liquid nitrogen. Then, DNA was extracted using a commercial kit and sequenced by 16S rDNA to verify the enhanced bacterial DNA extraction efficiency. Ultimately, we observed the following: (1) About 30 mg lyophilized meconium was the optimal amount for DNA extraction. (2) Freezing treatment for 6 h improved DNA extraction at −20°C. (3) DNA extraction efficiency was significantly higher with the immediate thaw strategy than with gradient thawing at −20°C, −80°C, and in liquid nitrogen. (4) Among the conditions of −20°C, −80°C, and liquid nitrogen, −20°C was the best freezing condition for both improving DNA extraction efficiency and preserving microbial species diversity in meconium, while liquid nitrogen was the worst condition. (5) Three freeze-thaw cycles could markedly enhance DNA extraction efficiency and preserve the species diversity of meconium microflora. We developed a feasible freeze-thaw pretreatment protocol to improve the extraction of microbial DNA from meconium, which may be beneficial for newborn bacterial colonization studies.

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