Optimising the detection of marine taxonomic richness using environmental DNA metabarcoding: the effects of filter material, pore size and extraction method

The analysis of environmental DNA (eDNA) using metabarcoding has increased in use as a method for tracking biodiversity of ecosystems. Little is known about eDNA in marine human-modified environments, such as commercial ports, which are key sites to monitor for anthropogenic impacts on coastal ecosystems. To optimise an eDNA metabarcoding protocol in these environments, seawater samples were collected in a commercial port and methodologies for concentrating and purifying eDNA were tested for their effect on eukaryotic DNA yield and subsequent richness of Operational Taxonomic Units (OTUs). Different filter materials [Cellulose Nitrate (CN) and Glass Fibre (GF)], with different pore sizes (0.5 µm, 0.7 µm and 1.2 µm) and three previously published liquid phase extraction methods were tested. The number of eukaryotic OTUs detected differed by a factor of three amongst the method combinations. The combination of CN filters with phenol-chloroform-isoamyl alcohol extractions recovered a higher amount of eukaryotic DNA and OTUs compared to GF filters and the chloroform-isoamyl alcohol extraction method. Pore size was not independent of filter material but did affect the yield of eukaryotic DNA. For the OTUs assigned to a highly successful non-indigenous species, Styelaclava, the two extraction methods with phenol significantly outperformed the extraction method without phenol; other experimental treatments did not contribute significantly to detection. These results highlight that careful consideration of methods is warranted because choice of filter material and extraction method create false negative detections of marine eukaryotic OTUs and underestimate taxonomic richness from environmental samples.

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Environmental DNA (eDNA) metabarcoding surveys extend the range of invasion for non-indigenous freshwater species in Eastern Europe.

10.1101/2021.05.16.444374 ◽

2021 ◽

Author(s):

Gert-Jan Jeunen ◽

Tatsiana Lipinskaya ◽

Helen Gajduchenko ◽

Viktoriya Golovenchik ◽

Michail Moroz ◽

...

Keyword(s):

Surface Water ◽

Water Samples ◽

False Negative ◽

Simultaneous Detection ◽

Environmental Dna ◽

Reference Database ◽

Indigenous Species ◽

Freshwater Species ◽

Traditional Approaches ◽

Surface Water Samples

Active environmental DNA (eDNA) surveillance through species-specific amplification has shown increased sensitivity in the detection of non-indigenous species (NIS) compared to traditional approaches. When many NIS are of interest, however, active surveillance decreases in cost- and time-efficiency. Passive surveillance through eDNA metabarcoding takes advantage of the complex DNA signal in environmental samples and facilitates the simultaneous detection of multiple species. While passive eDNA surveillance has previously detected NIS, comparative studies are essential to determine the ability of eDNA metabarcoding to accurately describe the range of invasion for multiple NIS versus alternative approaches. Here, we surveyed twelve sites, covering nine rivers across Belarus for NIS with three different techniques, i.e., an ichthyological, hydrobiological, and eDNA survey, whereby DNA was extracted from 500 mL surface water samples and amplified with two 16S rRNA primer assays targeting the fish and macro-invertebrate biodiversity. Nine non-indigenous fish and ten non-indigenous sediment-living macro-invertebrates were detected by traditional surveys, while seven NIS eDNA signals were picked up, including four fish, one aquatic and two sediment-living macro-invertebrates. Passive eDNA surveillance extended the range of invasion further north for two invasive fish and identified a new NIS for Belarus, the freshwater jellyfish Craspedacusta sowerbii. False-negative detections for the eDNA survey could be attributed to (i) preferential amplification of aquatic over sediment-living macro-invertebrates from surface water samples and (ii) an incomplete reference database. The evidence provided in this study recommends the implementation of both molecular-based and traditional approaches to maximize the probability of early detection of non-native organisms.

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Application of environmental DNA for monitoring and management of aquatic biological invasions: Emerging trends and advancements towards best practice

ARPHA Conference Abstracts ◽

10.3897/aca.4.e65476 ◽

2021 ◽

Vol 4 ◽

Author(s):

Emily Chen

Keyword(s):

Invasive Species ◽

Experimental Design ◽

Native Species ◽

Best Practice ◽

Environmental Dna ◽

Extraction Methods ◽

Indigenous Species ◽

Recent Emergence ◽

Species Specific ◽

Sampling Procedures

Introduction Aquatic Invasive Species (AIS) are a growing concern for global biodiversity as humans continue to accelerate the transport of non-indigenous species beyond their natural range. These species may possess traits that allow them to thrive in new environmental conditions such as non-selective feeding and high reproductive output, causing ecological harm through competition with native species for limited local resources. Consequently, environmental DNA (eDNA) has come to the forefront of AIS management in recent years as a promising method to detect or monitor invasive species using rapid and non-invasive sampling to complement traditional surveying. As eDNA’s potential is explored and beginning to be adopted for a variety of applications around the world, it is increasingly important to synthesize the trends in field and laboratory protocols from different working groups to establish guidelines that will allow greater comparability between studies and improve experimental design. Methodology and Results This meta-analytic study collated and reviewed information from previously published eDNA studies that targeted AIS in freshwater and marine environments to recognize current patterns in sampling techniques, laboratory protocols, and potential geographic or taxonomic biases. A total of 492 records from 192 full-text articles were used in the analysis, composed of 408 species-specific and 84 metabarcoding records. With regards to sampling procedures, many studies were not explicit enough for true replicability, lacking critical information such as the volume of filtered water and details of storage conditions. There was no observable trend for eDNA extraction methods in either species-specific or metabarcoding approaches, with choice of extraction method being mostly arbitrary among laboratories as well as influenced by the recent emergence of dedicated commercial kits . Discussion This analysis revealed a wide variety of choices for collecting and processing eDNA samples, so it is recommended that there should be some sort of standardized workflow diagram or decision tree for every stage of the experimental design in order for researchers to determine what approaches best meet their research objectives. There is also a clear need for improving metadata reporting guidelines; although the relevance of some criteria depends on the goals and limitations of specific projects, there should be a standardized minimum set of parameters to be reported for each eDNA study, from environmental variables to decontamination practices to PCR conditions. This will increase consistency and transparency through all stages of eDNA research, which is key to collectively improving methodologies and moving forward in this field.

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A comparison of different DNA extraction methods and molecular techniques for the detection and identification of foodborne pathogens

AIMS Microbiology ◽

10.3934/microbiol.2021019 ◽

2021 ◽

Vol 7 (3) ◽

pp. 304-319

Author(s):

Spyridon Andreas Papatheodorou ◽

◽

Panagiotis Halvatsiotis ◽

Dimitra Houhoula ◽

Keyword(s):

Dna Extraction ◽

Foodborne Pathogens ◽

Extraction Method ◽

Pathogenic Bacteria ◽

Low Cost ◽

Isoamyl Alcohol ◽

Extraction Methods ◽

Molecular Techniques ◽

Bacterial Dna ◽

Dna Extraction Method

<abstract> <p>Foodborne infections continue to plague Europe. Food safety monitoring is in crisis as the existing techniques for detecting pathogens do not keep up with the global rising of food production and consumption. Thus, the development of innovative techniques for detecting and identifying pathogenic bacteria has become critical. The aim of the present study was firstly to develop an innovative simple and low cost method of extracting bacterial DNA from contaminated food and water samples with <italic>Salmonella enteric</italic> subsp. <italic>enteric</italic> serovar Typhimurium and <italic>Listeria monocytogenes</italic> and its comparison with two commercial DNA extraction kits (Qiagen, Macherey-Nagel). Finally, pathogens' detection using two molecular techniques (PCR-electrophoresis, LAMP), in order to evaluate the best combination of DNA extraction and identification based on their sensitivity, cost, rapidity and simplicity. Considering the above criteria, among them, best was proved an in-house bacterial DNA extraction method, based on the chloroform-isoamyl alcohol protocol, with certain modifications. This technique showed statistically similar results in terms of sensitivity, compared to the commercial kits, while at the same time maintained high rapidity and much lower cost. Lastly, between the molecular techniques, LAMP was found more promising considering its simplicity, high rapidity and sensitivity. Conclusively, the in-house DNA extraction method along with the LAMP technique, was proven to be the best among the presented combinations.</p> </abstract>

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A pilot study of DNA yield from bloodstains on various surfaces using Phenol chloroform isoamyl alcohol (PCIA) and Chelex DNA extraction methods

GSC Biological and Pharmaceutical Sciences ◽

10.30574/gscbps.2020.13.2.0363 ◽

2020 ◽

Vol 13 (2) ◽

pp. 124-127

Author(s):

Ishwar Prasad Dubey ◽

R. K. Kumawat ◽

I. P. Tripathi ◽

Pankaj Shrivastava

Keyword(s):

Dna Extraction ◽

Extraction Method ◽

Short Tandem Repeats ◽

Tandem Repeats ◽

Isoamyl Alcohol ◽

Extraction Methods ◽

Forensic Dna ◽

Dna Yield ◽

Dna Extraction Methods ◽

Short Tandem

Presently, Short Tandem Repeats (STRs) based forensic DNA typing technology is being globally used in solving a diverse range of forensic cases such as paternity, identification of unknown dead bodies/skeletal remains, or suspect in a case of rape or mass rape. The technology has invaded its tentacles in almost all areas of criminal investigation in the last few decades. The present forensic DNA technology is based on capillary electrophoresis and utilizes short tandem repeats(STRs).On one hand, the technology is extensively used in the investigation of crime in highly sensitive cases, but on the another hand, obtaining DNA profile from forensic samples are highly challenging many times. Advent of PCR has been a boon for handling the challenging samples in forensic DNA analysis. The quality DNA profiles from challenging samples rely on the yield and quality of DNA, which is mainly dependent upon the method used for DNA extraction. Any specific method can never be thought of to be useful for all variety of samples. Still, Phenol Chloroform Isoamyl Alcohol (PCIA) organic extraction method has been proven to be useful for a wide variety of samples from the simplest saliva/blood to complex teeth and bone samples. In the present study, we compared the yield of DNA from blood stains recovered from various surfaces using the PCIA extraction method and Chelex DNA extraction methods and their compatibility with present-day STR based capillary electrophoresis typing. The mean value of DNA yield was found 50.5 ng/ µl and 32.25 ng/ µl by PCIA and Chelex DNA extraction methods, respectively. Overall, the highest yield was observed from all the tested samples from the PCIA method.

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Evaluation of DNA Extraction Methods Developed for Forensic and Ancient DNA Applications Using Bone Samples of Different Age

Genes ◽

10.3390/genes12020146 ◽

2021 ◽

Vol 12 (2) ◽

pp. 146

Author(s):

Catarina Xavier ◽

Mayra Eduardoff ◽

Barbara Bertoglio ◽

Christina Amory ◽

Cordula Berger ◽

...

Keyword(s):

Dna Extraction ◽

Ancient Dna ◽

Extraction Method ◽

Fragment Size ◽

Molecular Genetic ◽

Extraction Methods ◽

Degraded Dna ◽

Size Analysis ◽

Dna Fragments ◽

Dna Amount

The efficient extraction of DNA from challenging samples, such as bones, is critical for the success of downstream genotyping analysis in molecular genetic disciplines. Even though the ancient DNA community has developed several protocols targeting small DNA fragments that are typically present in decomposed or old specimens, only recently forensic geneticists have started to adopt those protocols. Here, we compare an ancient DNA extraction protocol (Dabney) with a bone extraction method (Loreille) typically used in forensics. Real-time quantitative PCR and forensically representative typing methods including fragment size analysis and sequencing were used to assess protocol performance. We used four bone samples of different age in replicates to study the effects of both extraction methods. Our results confirm Loreille’s overall increased gain of DNA when enough tissue is available and Dabney’s improved efficiency for retrieving shorter DNA fragments that is beneficial when highly degraded DNA is present. The results suggest that the choice of extraction method needs to be based on available sample, degradation state, and targeted genotyping method. We modified the Dabney protocol by pooling parallel lysates prior to purification to study gain and performance in single tube typing assays and found that up to six parallel lysates lead to an almost linear gain of extracted DNA. These data are promising for further forensic investigations as the adapted Dabney protocol combines increased sensitivity for degraded DNA with necessary total DNA amount for forensic applications.

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Impact of Extraction Method on the Detection of Quality Biomarkers in Normal vs. DFD Meat

Foods ◽

10.3390/foods10051097 ◽

2021 ◽

Vol 10 (5) ◽

pp. 1097

Author(s):

Laura González-Blanco ◽

Yolanda Diñeiro ◽

Andrea Díaz-Luis ◽

Ana Coto-Montes ◽

Mamen Oliván ◽

...

Keyword(s):

Extraction Method ◽

Protein Pattern ◽

Stress Protein ◽

Electrophoretic Pattern ◽

Extraction Methods ◽

Sds Page ◽

Weight Protein ◽

Triton X ◽

Protein Extractability ◽

Selection Of

The objective of this work was to demonstrate how the extraction method affects the reliability of biomarker detection and how this detection depends on the biomarker location within the cell compartment. Different extraction methods were used to study the sarcoplasmic and myofibrillar fractions of the Longissimus thoracis et lumborum muscle of young bulls of the Asturiana de los Valles breed in two quality grades, standard (Control) or dark, firm, and dry (DFD) meat. Protein extractability and the expression of some of the main meat quality biomarkers—oxidative status (lipoperoxidation (LPO) and catalase activity (CAT)), proteome (SDS-PAGE electrophoretic pattern), and cell stress protein (Hsp70)—were analyzed. In the sarcoplasmic fraction, buffers containing Triton X-100 showed significantly higher protein extractability, LPO, and higher intensity of high-molecular-weight protein bands, whereas the TES buffer was more sensitive to distinguishing differences in the protein pattern between the Control and DFD meat. In the myofibrillar fraction, samples extracted with the lysis buffer showed significantly higher protein extractability, whereas samples extracted with the non-denaturing buffer showed higher results for LPO, CAT, and Hsp70, and higher-intensity bands in the electrophoretic pattern. These findings highlight the need for the careful selection of the extraction method used to analyze the different biomarkers considering their cellular location to adapt the extractive process.

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Integrated Metabolomics and Transcriptomics Using an Optimised Dual Extraction Process to Study Human Brain Cancer Cells and Tissues

Metabolites ◽

10.3390/metabo11040240 ◽

2021 ◽

Vol 11 (4) ◽

pp. 240

Author(s):

Alison Woodward ◽

Alina Pandele ◽

Salah Abdelrazig ◽

Catherine A. Ortori ◽

Iqbal Khan ◽

...

Keyword(s):

Metabolic Pathways ◽

Extraction Method ◽

Limit Of Detection ◽

Rna Extraction ◽

Extraction Process ◽

Extraction Methods ◽

Serum Starvation ◽

Extraction Techniques ◽

Metabolic Genes ◽

Dual Extraction

The integration of untargeted metabolomics and transcriptomics from the same population of cells or tissue enhances the confidence in the identified metabolic pathways and understanding of the enzyme–metabolite relationship. Here, we optimised a simultaneous extraction method of metabolites/lipids and RNA from ependymoma cells (BXD-1425). Relative to established RNA (mirVana kit) or metabolite (sequential solvent addition and shaking) single extraction methods, four dual-extraction techniques were evaluated and compared (methanol:water:chloroform ratios): cryomill/mirVana (1:1:2); cryomill-wash/Econospin (5:1:2); rotation/phenol-chloroform (9:10:1); Sequential/mirVana (1:1:3). All methods extracted the same metabolites, yet rotation/phenol-chloroform did not extract lipids. Cryomill/mirVana and sequential/mirVana recovered the highest amounts of RNA, at 70 and 68% of that recovered with mirVana kit alone. sequential/mirVana, involving RNA extraction from the interphase of our established sequential solvent addition and shaking metabolomics-lipidomics extraction method, was the most efficient approach overall. Sequential/mirVana was applied to study a) the biological effect caused by acute serum starvation in BXD-1425 cells and b) primary ependymoma tumour tissue. We found (a) 64 differentially abundant metabolites and 28 differentially expressed metabolic genes, discovering four gene-metabolite interactions, and (b) all metabolites and 62% lipids were above the limit of detection, and RNA yield was sufficient for transcriptomics, in just 10 mg of tissue.

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A Structural Analysis Based Feature Extraction Method for OCR System For Myanmar Printed Document Images

International Journal of Computer Vision and Image Processing ◽

10.4018/ijcvip.2012010102 ◽

2012 ◽

Vol 2 (1) ◽

pp. 16-41 ◽

Cited By ~ 1

Author(s):

Htwe Pa Pa Win ◽

Phyo Thu Thu Khine ◽

Khin Nwe Ni Tun

Keyword(s):

Feature Extraction ◽

Structural Analysis ◽

Character Recognition ◽

Optical Character Recognition ◽

Extraction Method ◽

Recognition Performance ◽

Extraction Methods ◽

Support Vector ◽

Svm Classifier ◽

Feature Extraction Method

This paper proposes a new feature extraction method for off-line recognition of Myanmar printed documents. One of the most important factors to achieve high recognition performance in Optical Character Recognition (OCR) system is the selection of the feature extraction methods. Different types of existing OCR systems used various feature extraction methods because of the diversity of the scripts’ natures. One major contribution of the work in this paper is the design of logically rigorous coding based features. To show the effectiveness of the proposed method, this paper assumed the documents are successfully segmented into characters and extracted features from these isolated Myanmar characters. These features are extracted using structural analysis of the Myanmar scripts. The experimental results have been carried out using the Support Vector Machine (SVM) classifier and compare the pervious proposed feature extraction method.

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Compositional Analysis and Aroma Evaluation of Feijoa Essential Oils from New Zealand Grown Cultivars

Molecules ◽

10.3390/molecules24112053 ◽

2019 ◽

Vol 24 (11) ◽

pp. 2053 ◽

Cited By ~ 2

Author(s):

Yaoyao Peng ◽

Karen Suzanne Bishop ◽

Siew Young Quek

Keyword(s):

New Zealand ◽

Essential Oil ◽

Essential Oils ◽

Extraction Method ◽

Compositional Analysis ◽

Extraction Methods ◽

Essential Oil Composition ◽

Oil Composition ◽

Active Compounds ◽

Initial Comparison

Feijoa is an aromatic fruit and the essential oil from feijoa peel could be a valuable by-product in the juicing industry. An initial comparison of the essential oil extraction methods, steam-distillation and hydro-distillation, was conducted. The volatile compounds in the essential oils from four feijoa cultivars were identified and semi-quantified by GC-MS and the aroma active compounds in each essential oil were characterized using SPME-GC-O-MS. Hydro-distillation, with a material to water ratio of 1:4 and an extraction time of 90 min, was the optimized extraction method for feijoa essential oil. The Wiki Tu cultivar produced the highest essential oil yield among the four selected cultivars. A total of 160 compounds were detected, among which 90 compounds were reported for the first time in feijoa essential oils. Terpenes and esters were dominant compounds in feijoa essential oil composition and were also major contributors to feijoa essential oil aroma. Key aroma active compounds in feijoa essential oils were α-terpineol, ethyl benzoate, (Z)-3-hexenyl hexanoate, linalool, (E)-geraniol, 2-undecanone, 3-octanone, α-cubebene, and germacrene D. This is the first report on the optimization of the extraction method and the establishment of the aroma profile of feijoa essential oils, with a comparison of four New Zealand grown cultivars.

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Optimization of Protein Extraction Method for 2DE Proteomics of Goat’s Milk

Molecules ◽

10.3390/molecules25112625 ◽

2020 ◽

Vol 25 (11) ◽

pp. 2625

Author(s):

Muzammeer Mansor ◽

Jameel R. Al-Obaidi ◽

Nurain Nadiah Jaafar ◽

Intan Hakimah Ismail ◽

Atiqah Farah Zakaria ◽

...

Keyword(s):

Extraction Method ◽

Protein Extraction ◽

Chloroform Solution ◽

Milk Proteins ◽

Peptide Mass Fingerprinting ◽

Extraction Methods ◽

Dairy Goats ◽

Goat’S Milk ◽

Peptide Mass ◽

Goat's Milk

Two-dimensional electrophoretic (2DE)-based proteomics remains a powerful tool for allergenomic analysis of goat’s milk but requires effective extraction of proteins to accurately profile the overall causative allergens. However, there are several current issues with goat’s milk allergenomic analysis, and among these are the absence of established standardized extraction method for goat’s milk proteomes and the complexity of goat’s milk matrix that may hamper the efficacy of protein extraction. This study aimed to evaluate the efficacies of three different protein extraction methods, qualitatively and quantitatively, for the 2DE-proteomics, using milk from two commercial dairy goats in Malaysia, Saanen, and Jamnapari. Goat’s milk samples from both breeds were extracted by using three different methods: a milk dilution in urea/thiourea based buffer (Method A), a triphasic separation protocol in methanol/chloroform solution (Method B), and a dilution in sulfite-based buffer (Method C). The efficacies of the extraction methods were assessed further by performing the protein concentration assay and 1D and 2D SDS-PAGE profiling, as well as identifying proteins by MALDI-TOF/TOF MS/MS. The results showed that method A recovered the highest amount of proteins (72.68% for Saanen and 71.25% for Jamnapari) and produced the highest number of protein spots (199 ± 16.1 and 267 ± 10.6 total spots for Saanen and Jamnapari, respectively) with superior gel resolution and minimal streaking. Six milk protein spots from both breeds were identified based on the positive peptide mass fingerprinting matches with ruminant milk proteins from public databases, using the Mascot software. These results attest to the fitness of the optimized protein extraction protocol, method A, for 2DE proteomic and future allergenomic analysis of the goat’s milk.

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