scholarly journals Taxonomic identification of airborne pollen from complex environmental samples by DNA metabarcoding: a methodological study for optimizing protocols

2017 ◽  
Author(s):  
Kleopatra Leontidou ◽  
Cristiano Vernesi ◽  
Johannes De Groeve ◽  
Fabiana Cristofolini ◽  
Despoina Vokou ◽  
...  
Keyword(s):  
Sample Preparation ◽  
Chloroplast Dna ◽  
Dna Extraction ◽  
Environmental Samples ◽  
Airborne Pollen ◽  
Reference Database ◽  
Taxonomic Assignment ◽  
Spore Trap ◽  
Custom Made ◽  
Dna Metabarcoding

AbstractMetabarcoding is a promising DNA-based method for identifying airborne pollen from environmental samples with advantages over microscopic methods. This method requires several preparatory steps of the samples, with the extraction protocol being of fundamental importance to obtain an optimal DNA yield. Currently, there is no consensus in sample preparation and DNA extraction, especially for gravimetric pollen samplers. Therefore, the aim of this study was to develop protocols to process environmental samples for pollen DNA extraction and further metabarcoding analysis, and to assess the efficacy of these protocols for the taxonomic assignment of airborne pollen, collected by gravimetric (Tauber trap) and volumetric samplers (Burkard spore trap). Protocols were tested across an increasing complexity of samples, from single-species pure pollen to environmental samples. A short fragment (about 150 base pair) of chloroplast DNA was amplified by universal primers for plants (trnL). After PCR amplification, amplicons were Sanger-sequenced and taxonomic assignment was accomplished by comparison to a custom-made reference database including chloroplast DNA sequences of 46 plant families, including most of the anemophilous taxa occurring in the study area (Trentino, Italy, Eastern Italian Alps). Using as a benchmark the classical morphological pollen analysis, it emerged that DNA metabarcoding is applicable efficiently across a complexity of samples, provided that sample preparation, DNA extraction and amplification protocols are specifically optimized.

scholarly journals Rapid Filtration Separation-Based Sample Preparation Method for Bacillus Spores in Powdery and Environmental Matrices

2011 ◽  
Vol 78 (5) ◽  
pp. 1505-1512 ◽  
Author(s):  
Sandra Isabel ◽  
Maurice Boissinot ◽  
Isabelle Charlebois ◽  
Chantal M. Fauvel ◽  
Lu-E Shi ◽  
...  
Keyword(s):  
Sample Preparation ◽  
Dna Extraction ◽  
Environmental Samples ◽  
Extraction Procedure ◽  
Pcr Detection ◽  
Filter Method ◽  
Nucleic Acid Detection ◽  
Quality Level ◽  
Content Type ◽  
Bacillus Spores

ABSTRACTAuthorities frequently need to analyze suspicious powders and other samples for biothreat agents in order to assess environmental safety. Numerous nucleic acid detection technologies have been developed to detect and identify biowarfare agents in a timely fashion. The extraction of microbial nucleic acids from a wide variety of powdery and environmental samples to obtain a quality level adequate for these technologies still remains a technical challenge. We aimed to develop a rapid and versatile method of separating bacteria from these samples and then extracting their microbial DNA.Bacillus atrophaeussubsp.globigiiwas used as a simulant ofBacillus anthracis. We studied the effects of a broad variety of powdery and environmental samples on PCR detection and the steps required to alleviate their interference. With a benchmark DNA extraction procedure, 17 of the 23 samples investigated interfered with bacterial lysis and/or PCR-based detection. Therefore, we developed thedual-filter method forappliedrecovery of microbial particles fromenvironmental and powdery samples (DARE). The DARE procedure allows the separation of bacteria from contaminating matrices that interfere with PCR detection. This procedure required only 2 min, while the DNA extraction process lasted 7 min, for a total of <10 min. This sample preparation procedure allowed the recovery of cleaned bacterial spores and relieved detection interference caused by a wide variety of samples. Our procedure was easily completed in a laboratory facility and is amenable to field application and automation.

scholarly journals A new oomycete metabarcoding method using the rps10 gene

2021 ◽  
Author(s):  
Zachary S. L. Foster ◽  
Felipe E Albornoz ◽  
Valerie J Fieland ◽  
Meredith M Larsen ◽  
Frank Andrew Jones ◽  
...  
Keyword(s):  
Environmental Samples ◽  
Illumina Miseq ◽  
Taxonomic Resolution ◽  
Reference Database ◽  
Mock Community ◽  
Operational Taxonomic Units ◽  
Wide Range ◽  
Dna Metabarcoding ◽  
Improved Methods

Oomycetes are a group of eukaryotes related to brown algae and diatoms, many of which cause diseases in plants and animals. Improved methods are needed for rapid and accurate characterization of oomycete communities using DNA metabarcoding. We have identified the mitochondrial 40S ribosomal protein S10 gene (rps10) as a locus useful for oomycete metabarcoding and provide primers predicted to amplify all oomycetes based on available reference sequences from a wide range of taxa. We evaluated its utility relative to a popular barcode, the internal transcribed spacer 1 (ITS1), by sequencing environmental samples and a mock community using Illumina MiSeq. Amplified sequence variants (ASVs) and operational taxonomic units (OTUs) were identified per community. Both the sequence and predicted taxonomy of ASVs and OTUs were compared to the known composition of the mock community. Both rps10 and ITS yielded ASVs with sequences matching 21 of the 24 species in the mock community and matching all 24 when allowing for a 1 bp difference. Taxonomic classifications of ASVs included 23 members of the mock community for rps10 and 17 for ITS1. Sequencing results for the environmental samples suggest the proposed rps10 locus results in substantially less amplification of non-target organisms than the ITS1 method. The amplified rps10 region also has higher taxonomic resolution than ITS1, allowing for greater discrimination of closely related species. We present a new website with a searchable rps10 reference database for species identification and all protocols needed for oomycete metabarcoding. The rps10 barcode and methods described herein provide an effective tool for metabarcoding oomycetes using short-read sequencing.

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 DNA metabarcoding of airborne pollen: new protocols for improved taxonomic identification of environmental samples

Aerobiologia ◽  
2017 ◽  
Vol 34 (1) ◽  
pp. 63-74 ◽  
Author(s):  
Kleopatra Leontidou ◽  
Cristiano Vernesi ◽  
Johannes De Groeve ◽  
Fabiana Cristofolini ◽  
Despoina Vokou ◽  
...  
Keyword(s):  
Environmental Samples ◽  
Airborne Pollen ◽  
Taxonomic Identification ◽  
Dna Metabarcoding

Pollutant Identification by Selected Area Electron Diffraction

1974 ◽  
Vol 32 ◽  
pp. 532-533
Author(s):  
R. E. Ferrell ◽  
G. G. Paulson ◽  
C. W. Walker
Keyword(s):  
Thermal Treatment ◽  
Electron Diffraction ◽  
Sample Preparation ◽  
Crystal Structures ◽  
Water Samples ◽  
Environmental Samples ◽  
Short Review ◽  
Selected Area Electron Diffraction ◽  
Multiple Component

Selected area electron diffraction (SAD) has been used successfully to determine crystal structures, identify traces of minerals in rocks, and characterize the phases formed during thermal treatment of micron-sized particles. There is an increased interest in the method because it has the potential capability of identifying micron-sized pollutants in air and water samples. This paper is a short review of the theory behind SAD and a discussion of the sample preparation employed for the analysis of multiple component environmental samples.

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 Methodological Approaches to DNA Authentication of Foods, Wines and Raw Materials for Their Production

Foods ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 595
Author(s):  
Aram G. Galstyan ◽  
Vladislav K. Semipyatniy ◽  
Irina Yu. Mikhailova ◽  
Khamid Kh. Gilmanov ◽  
Alana V. Bigaeva ◽  
...  
Keyword(s):  
Sample Preparation ◽  
Dna Extraction ◽  
Raw Materials ◽  
Direct Sequencing ◽  
Genetic Identification ◽  
Proteinase K ◽  
Vitis Vinifera L ◽  
Acid Extraction ◽  
Plant Component ◽  
Sensitivity Specificity

DNA authentication of wines is a process of verifying their authenticity by genetic identification of the main plant component. The sample preparation of experimental and commercial wines was carried out by precipitation of wine debris by centrifugation with preliminary exposure with precipitators and co-precipitators, including developed macro- and micro- volume methods applicable to white or red wines, using polyvinylpyrrolidone as a co-precipitator. Addition of 2-mercaptoethanol and proteinase K to the lysing solution made it possible to adapt the technology for DNA extraction from the precipitated wine debris. The additionally tested technique of DNA extraction from wine debris by dimethyl sulfoxide (DMSO) lysis had fewer stages and, consequently, a lower risk of contamination. The results of further testing of one of the designed primer pairs (UFGT-F1 and UFGT-R1) in conjunction with the tested methods of wine material sample preparation and nucleic acid extraction, showed the advantage in the given set of oligonucleotides over previously used ones in terms of sensitivity, specificity and reproducibility. The developing strategy for genetic identification of grape varieties and DNA authentication of wines produced from them based on direct sequencing of polymerase chain reaction (PCR) products is implemented by interpreting the detected polymorphic positions of variable Vitis vinifera L. UFGT gene locus with distribution and split into 13 UFGT gene-associated groups.

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