scholarly journals Metabarcoding for the parallel identification of several hundred predators and their preys: application to bat species diet analysis

2017 ◽  
Author(s):  
Maxime Galan ◽  
Jean-Baptiste Pons ◽  
Orianne Tournayre ◽  
Éric Pierre ◽  
Maxime Leuchtmann ◽  
...  
Keyword(s):  
Cost Effective ◽  
Extraction Methods ◽  
False Positives ◽  
Diet Analysis ◽  
Taxonomic Resolution ◽  
Conservation Strategies ◽  
Faecal Pellets ◽  
Diet Variability ◽  
Dna Extraction Methods ◽  
Mock Communities

AbstractAssessing diet variability is of main importance to better understand the biology of bats and design conservation strategies. Although the advent of metabarcoding has facilitated such analyses, this approach does not come without challenges. Biases may occur throughout the whole experiment, from fieldwork to biostatistics, resulting in the detection of false negatives, false positives or low taxonomic resolution. We detail a rigorous metabarcoding approach based on a short COI minibarcode and two-step PCR protocol enabling the ‘all at once’ taxonomic identification of bats and their arthropod preys for several hundreds of samples. Our study includes faecal pellets collected in France from 357 bats representing 16 species, as well as insect mock communities that mimic bat meals of known composition, negative and positive controls. All samples were analysed using three replicates. We compare the efficiency of DNA extraction methods and we evaluate the effectiveness of our protocol using identification success, taxonomic resolution, sensitivity, and amplification biases. Our parallel identification strategy of predators and preys reduces the risk of mis-assigning preys to wrong predators and decreases the number of molecular steps. Controls and replicates enable to filter the data and limit the risk of false positives, hence guaranteeing high confidence results for both prey occurrence and bat species identification. We validate 551 COI variants from arthropod including 18 orders, 117 family, 282 genus and 290 species. Our method therefore provides a rapid, resolutive and cost-effective screening tool for addressing evolutionary ecological issues or developing ‘chirosurveillance’ and conservation strategies.

scholarly journals Macroinvertebrate community assessment and biomonitoring of European water bodies. Is a multimarker approach necessary?

2021 ◽  
Vol 4 ◽  
Author(s):  
Juan Antonio Villaescusa ◽  
Mª José Villena ◽  
Raquel González ◽  
Antonio Picazo ◽  
Verónica Rojo ◽  
...  
Keyword(s):  
Ecological Status ◽  
Extraction Methods ◽  
Macroinvertebrate Community ◽  
Universal Primers ◽  
Community Assessment ◽  
Biogeographic Regions ◽  
And Performance ◽  
Dna Extraction Methods ◽  
Taxonomic Groups ◽  
Mock Communities

The present work, included in the European Project BIOWAT, aims to develop and validate the use of genomic tools or metabarcoding for its utilization as a routine method for river biomonitoring in different European Biogeographical regions. The project included sampling points in three biogeographic regions, Mediterranean (Spain), Continental (Germany) and Boreal (Finland). The current development of the study was designed using mock communities obtained from the three mentioned areas and different aspects were tested: DNA extraction methods, selection of informative region (16S vs COI), design and performance of primers, bioinformatic pipeline, etc… Although the use of COI has become very popular, and its barcode database is more complete, the use of mitochondrial 16S as taxonomic marker can provide similar or even better results when accompanied by a rich local barcode database Elbrecht 2016. In this presentation, the results and conclusions obtained for the biomonitoring of nine rivers (3 for each of the biogeographic regions) using 16S as DNA marker and a local barcode database are shown. The results of ecological status assignment using 16S marker were promising, showing a good correlation between morphological determinations and metabarcoding data for most of the studied rivers. However, in some cases, the metabarcoding data showed a jump in the ecological status class (to better or worst status), highlighting the existence of some problems related with primers (for some taxonomic groups) or missing taxa in the barcode database that still need to be solved prior the utilization of this method on a routine basis. Additionally, for the studied Mediterranean rivers, a complementary analysis using COI as marker was made, using the universal primers developed by Elbrecht and Leese 2017. In general, this marker showed better results in the identification for some taxa, whereas other included in the mock communities were not identified showing important problems that could be related with primers (sometimes not well covering characteristic taxa present in other biogeographic regions) or the lack of a complete COI macroinvertebrate barcode databases in the Iberian Peninsula for the case of Spain Múrria 2020. Our results show that both markers have the potential to produce a good identification of benthic macroinvertebrates, showing an acceptable correlation between morphology and metabarcoding approaches. However, none of them is able to amplify all of the present groups, so the parallel use of both markers (mitochondrial 16S and COI) in a multimarker approach could solve some of the problems, giving a more complete profile of the macroinvertebrate community. This approach has already been proposed and can lead the future of macroinvertebrate community assessment Ficetola 2020, Martins 2020.

scholarly journals A Simple, Cost-Effective, and Automation-Friendly Direct PCR Approach for Bacterial Community Analysis

mSystems ◽  
2021 ◽  
Author(s):  
Fangchao Song ◽  
Jennifer V. Kuehl ◽  
Arjun Chandran ◽  
Adam P. Arkin
Keyword(s):  
Cost Effective ◽  
Community Analysis ◽  
Extraction Methods ◽  
Dna Purification ◽  
Purification Step ◽  
Direct Pcr ◽  
Rrna Sequencing ◽  
Dna Loss ◽  
Dna Extraction Methods ◽  
Low Cell Density

Understanding bacterial interactions and assembly in complex microbial communities using 16S rRNA sequencing normally requires a large experimental load. However, the current DNA extraction methods, including cell disruption and genomic DNA purification, are normally biased, costly, time-consuming, labor-intensive, and not amenable to miniaturization by droplets or 1,536-well plates due to the significant DNA loss during the purification step for tiny-volume and low-cell-density samples.

scholarly journals Evaluation of a modified salt-out method for DNA extraction from whole blood lymphocytes: A simple and economical method for gene polymorphism

2018 ◽  
Author(s):  
Mohammad Shokrzadeh ◽  
Abbas Mohammadpour
Keyword(s):  
Gene Polymorphism ◽  
Dna Extraction ◽  
Ethylenediaminetetraacetic Acid ◽  
Genetic Research ◽  
Cost Effective ◽  
Extraction Methods ◽  
Economical Method ◽  
Modified Method ◽  
Dna Extraction Methods ◽  
And Control

  Extraction of high-quality and-quantity DNA is a fundamental requirement for genetic research. It is very important to address the use of DNA extraction methods that are simple and cost-effective in gene polymorphism with large number of samples. This study was designed to investigate the optimal DNA extraction from lymphocytic cells by salt-out method. In this study, 200 blood samples of the two groups of patients and control were collected and transferred to Ethylenediaminetetraacetic acid-containing tubes. Afterwards, DNA was extracted from 1 ml of blood cells by modified salt-out method. Furthermore, three parameters in this research were evaluated, including quality (optimal density at 260 nm), quantity (DNA concentration) by electrophoresis, and efficiency of extracted DNA or polymerase chain reaction (PCR) status. The findings revealed that extracted DNA had excellent concentration and purity. The obtained results of electrophoresis confirmed the absence of any fragments in the extracted DNA. The PCR of the extracted DNA were successful, indicating lack of inhibitors in the reaction. According to the results of this study, this modified method can be used as a simple, efficient, and economical method for DNA extraction.

scholarly journals Evaluation of DNA Extraction Methods and Bioinformatic Pipelines for Marine Nano- and Pico-Eukaryotic Plankton Analysis

2021 ◽  
Vol 7 ◽  
Author(s):  
Marta Muñoz-Colmenero ◽  
Ana Sánchez ◽  
Begoña Correa ◽  
Francisco G. Figueiras ◽  
Jose L. Garrido ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Environmental Sample ◽  
Environmental Samples ◽  
Extraction Methods ◽  
Ion Torrent ◽  
Taxonomic Assignment ◽  
Dna Quality ◽  
Eukaryotic Species ◽  
Dna Extraction Methods ◽  
Mock Communities

The smallest size fractions of plankton, nano- and pico-plankton, have been highlighted due to they accomplish key functions in marine ecosystems. However, the knowledge about some of them is scarce because they are difficult or impossible to be detected and identified with non-DNA-based methodologies. Here we have evaluated five DNA extraction protocols (MT1–MT5) and seven bioinformatic pipelines (P1–P7) to find the best protocol for detecting most of the eukaryotic species of nano- and pico-plankton present in an environmental sample using Ion Torrent technology. The protocol MT3 was the most reproducible methodology, showing less variation among samples, good DNA quality and sufficient quantity to amplify and sequence the eukaryote species, offering the best results after sequencing. For bioinformatic analyses, P1 and P7 resulted in the highest percentage of detection for the difficult-to-detect species in mock communities. However, only P1 avoided the confusion with other closed species during the taxonomic assignment. The final protocols, MT3-P1 (free) and MT3-P7 (private), showed good and consistent results when they were applied to an environmental sample, being a valuable tool to study the eukaryotes present in environmental samples of nano- and pico-plankton, even for the genera that are difficult to be detected by other techniques.

OPTIMIZATION OF DNA EXTRACTION METHODS FROM BIOLOGICAL MATERIALS OF HONEY BEES IN A DIFFERENT METAMOTPHOSIS PHASE

2016 ◽  
Vol 52 ◽  
pp. 171-176
Author(s):  
M. Palkina ◽  
O. Metlitska
Keyword(s):  
Ion Exchange ◽  
Dna Extraction ◽  
Honey Bees ◽  
Biological Material ◽  
Exchange Resin ◽  
Ion Exchange Resin ◽  
Extraction Methods ◽  
Chelex 100 ◽  
Drone Brood ◽  
Dna Extraction Methods

The aim of the research – adaptation, optimization and using of existing DNA extraction methods from bees’ biological material with the reagent «Chelex-100" under complex economic conditions of native laboratories, which will optimize labour costs and improve the economic performance of DNA extraction protocol. Materials and methods. In order to conduct the research the samples of honey bees’ biological material: queen pupae exuviae, larvae of drone brood, some adult bees’ bodies (head and thorax) were selected. Bowl and drone brood were obtained from the experimental bee hives of Institute of Apiculture nd. a. P. I. Prokopovich of NAAS. DNA extraction from biosamples of Apis mellifera ssp. was carried out using «Chelex-100®» ion exchange resin in different concentrations and combinations. Before setting tests for determination of quantitative and quality indexes, dilution of DNA samples of the probed object was conducted in ratio 1:40. The degree of contamination with protein and polysaccharide fractions (OD 260/230), quantitative content of DNA (OD 260/280) in the extracted tests were conducted using spectrophotometer of «Biospec – nano» at the terms of sample volume in 2 µl and length of optical way in 0,7 mm [7]. Verification of DNA samples from biological material of bees, isolated by «Chelex-100®», was conducted after cold keeping during 24 hours at 20°C using PСR with primaries to the fragment of gene of quantitative trait locus (QTL) Sting-2 of next structure [8]:  3' – CTC GAC GAG ACG ACC AAC TTG – 5’; 3' – AAC CAG AGT ATC GCG AGT GTT AC – 5’ Program of amplification: 94 °C – 5 minutes – 1 cycle; 94 °C – 1 minute, 57°C – 1 minute, 72 °C – 2 minutes – 30 cycles; elongation after 72°C during 2 minutes – 1 cycle. The division of obtained amplicons was conducted by gel electrophoresis at a low current – 7 µÀ, in 1,5 % agarose gel (Sigma ®) in TAE buffer [7]. The results. At the time of optimization of DNA isolation methods, according to existing methods of foreign experts, it was found optimal volume of ion exchange resin solution was in the proposed concentration: instead of 60 µl of solution used 120 µl of «Chelex-100®», time of incubation was also amended from 30 minutes to 180 minutes [9]. The use of the author's combination of method «Chelex-100®» with lysis enzymes, proteinase K and detergents (1M dithiothreitol), as time of incubation was also amended, which was reduced to 180 minutes instead of the proposed 12 hours [10]. Changes in quality characteristics of obtained DNA in samples after reduction in incubation time were not found. Conclusions. The most economical method of DNA isolation from bees’ biological material is 20% solution of «Chelex-100» ion exchange resin with the duration of the incubation period of 180 minutes. It should also be noted that the best results can be obtained from exuviae, selected immediately after the queen’s exit from bowl, that reduces the likelihood of DNA molecules destruction under the influence of nucleases activation, but not later than 12 hours from release using the technology of isolated obtain of queens.

scholarly journals Impact of Collection Volume and DNA Extraction Method on the Detection of Biomarkers and HPV DNA in First-Void Urine

Molecules ◽  
2021 ◽  
Vol 26 (7) ◽  
pp. 1989
Author(s):  
Laura Téblick ◽  
Severien Van Keer ◽  
Annemie De Smet ◽  
Pierre Van Damme ◽  
Michelle Laeremans ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Internal Control ◽  
Extraction Methods ◽  
Urine Collection ◽  
Hpv Dna ◽  
Non Invasive ◽  
Herpesvirus 1 ◽  
Dna Extraction Methods ◽  
Detection Of Biomarkers ◽  
High Risk Hpv

The potential of first-void (FV) urine as a non-invasive liquid biopsy for detection of human papillomavirus (HPV) DNA and other biomarkers has been increasingly recognized over the past decade. In this study, we investigated whether the volume of this initial urine stream has an impact on the analytical performance of biomarkers. In parallel, we evaluated different DNA extraction protocols and introduced an internal control in the urine preservative. Twenty-five women, diagnosed with high-risk HPV, provided three home-collected FV urine samples using three FV urine collection devices (Colli-Pee) with collector tubes that differ in volume (4, 10, 20 mL). Each collector tube was prefilled with Urine Conservation Medium spiked with phocine herpesvirus 1 (PhHV-1) DNA as internal control. Five different DNA extraction protocols were compared, followed by PCR for GAPDH and PhHV-1 (qPCR), HPV DNA, and HBB (HPV-Risk Assay), and ACTB (methylation-specific qPCR). Results showed limited effects of collection volume on human and HPV DNA endpoints. In contrast, significant variations in yield for human endpoints were observed for different DNA extraction methods (p < 0.05). Additionally, the potential of PhHV-1 as internal control to monitor FV urine collection, storage, and processing was demonstrated.

scholarly journals A Rapid Bacteriophage DNA Extraction Method

2018 ◽  
Vol 1 (3) ◽  
pp. 27 ◽  
Author(s):  
Džiuginta Jakočiūnė ◽  
Arshnee Moodley
Keyword(s):  
Dna Extraction ◽  
Illumina Miseq ◽  
Extraction Methods ◽  
Resistant Bacteria ◽  
Simple Method ◽  
Antibiotic Resistant ◽  
Phage Dna ◽  
Specialized Equipment ◽  
Miseq Platform ◽  
Dna Extraction Methods

Bacteriophages (phages) are intensely investigated as non-antibiotic alternatives to circumvent antibiotic resistance development as well as last resort therapeutic options against antibiotic resistant bacteria. As part of gaining a better understanding of phages and to determine if phages harbor putative virulence factors, whole genome sequencing is used, for which good quality phage DNA is needed. Traditional phage DNA extraction methods are tedious and time consuming, requiring specialized equipment e.g., an ultra-centrifuge. Here, we describe a quick and simple method (under four hours) to extract DNA from double stranded DNA (dsDNA) phages at titers above 1.0 × 1010 plaque-forming units (PFU)/mL. This DNA was suitable for library preparation using the Nextera XT kit and sequencing on the Illumina MiSeq platform.

131-P: Protein contamination in manual DNA extraction methods

2007 ◽  
Vol 68 (1) ◽  
pp. S80
Author(s):  
Victoriano J. Leon ◽  
Alberto J. Leon ◽  
Juan Luis Garcia
Keyword(s):  
Dna Extraction ◽  
Extraction Methods ◽  
P Protein ◽  
Protein Contamination ◽  
Dna Extraction Methods
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