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 Can non-destructive DNA extraction of bulk invertebrate samples be used for metabarcoding?

PeerJ ◽  
2018 ◽  
Vol 6 ◽  
pp. e4980 ◽  
Author(s):  
Melissa E. Carew ◽  
Rhys A. Coleman ◽  
Ary A. Hoffmann
Keyword(s):  
Dna Extraction ◽  
Dna Barcode ◽  
Extraction Methods ◽  
Biological Communities ◽  
Freshwater Invertebrate ◽  
High Throughput Dna Sequencing ◽  
Dna Extraction Methods ◽  
Taxonomic Groups ◽  
Taxonomic Studies ◽  
Non Destructive

Background High throughput DNA sequencing of bulk invertebrate samples or metabarcoding is becoming increasingly used to provide profiles of biological communities for environmental monitoring. As metabarcoding becomes more widely applied, new reference DNA barcodes linked to individual specimens identified by taxonomists are needed. This can be achieved through using DNA extraction methods that are not only suitable for metabarcoding but also for building reference DNA barcode libraries. Methods In this study, we test the suitability of a rapid non-destructive DNA extraction method for metabarcoding of freshwater invertebrate samples. Results This method resulted in detection of taxa from many taxonomic groups, comparable to results obtained with two other tissue-based extraction methods. Most taxa could also be successfully used for subsequent individual-based DNA barcoding and taxonomic identification. The method was successfully applied to field-collected invertebrate samples stored for taxonomic studies in 70% ethanol at room temperature, a commonly used storage method for freshwater samples. Discussion With further refinement and testing, non-destructive extraction has the potential to rapidly characterise species biodiversity in invertebrate samples, while preserving specimens for taxonomic investigation.

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.

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.

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.

Initiation, Development and Anatomy of Lignotubers in Some Species of Eucalyptus

1984 ◽  
Vol 32 (4) ◽  
pp. 415 ◽  
Author(s):  
DJ Carr ◽  
R Jahnke ◽  
SGM Carr
Keyword(s):  
Analytical Study ◽  
Growth Habit ◽  
Stem Growth ◽  
Morphological Development ◽  
Alternative Pathways ◽  
Stem Wood ◽  
Initial Survey ◽  
And Performance ◽  
Lateral Fusion ◽  
Taxonomic Groups

An initial survey of the diversity of early lignotuber development in Eucalyptus and an analytical study of the anatomy of young lignotubers and the seedling stem are presented. Studies of the early stages of the morphological development of the lignotuber in 13 species, representative of five taxonomic groups, resulted in the recognition of four modes of lignotuber initiation. The importance to lignotuber formation of the presence of a suite of accessory buds, adaxial to the axillary bud, is emphasized but lignotuber initiation is not in all cases associated with these buds. Lignotuber buds are derived by branching from existing buds, ultimately from the accessory buds of the node. Following its initiation, the possibilities of later morphological development of the lignotuber are discussed. Lignotuber growth may dominate over stem growth and the lignotubers at a node may then fuse laterally to encircle the stem. Stem growth, on the other hand, may dominate over lignotuber growth and the lignotuber then appears to regress. The consequences for the growth habit of the plant of these alternative pathways of development are outlined. The wood of young lignotubers (and that of the swollen hypocotyl) is shown to be different in composition and in the sizes of its elements from that of seedling stem wood; these differences owe their origin to differences in the nature and performance of the cambia of the lignotuber and stem. In lateral fusion of the lignotubers at a node, and their upward and downwards extension over the stem, e.g. over the hypocotyl, stem cambial initials are either progressively lost or, more likely, converted to lignotuber-type initials. The possibility of the reverse process occumng in stem dominance is discussed.

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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