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

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 PIMBA: a PIpeline for MetaBarcoding Analysis

2021 ◽  
Author(s):  
Renato R. M. Oliveira ◽  
Raissa L S Silva ◽  
Gisele L. Nunes ◽  
Guilherme Oliveira
Keyword(s):  
Environmental Samples ◽  
Next Generation Sequencing Data ◽  
Ecological Studies ◽  
Sequencing Data ◽  
Monitoring Method ◽  
Computational Tools ◽  
Dna Metabarcoding ◽  
Reference Databases ◽  
User Friendly ◽  
Generation Sequencing

DNA metabarcoding is an emerging monitoring method capable of assessing biodiversity from environmental samples (eDNA). Advances in computational tools have been required due to the increase of Next-Generation Sequencing data. Tools for DNA metabarcoding analysis, such as MOTHUR, QIIME, Obitools, and mBRAVE have been widely used in ecological studies. However, some difficulties are encountered when there is a need to use custom databases. Here we present PIMBA, a PIpeline for MetaBarcoding Analysis, which allows the use of customized databases, as well as other reference databases used by the softwares mentioned here. PIMBA is an open-source and user-friendly pipeline that consolidates all analyses in just three command lines.

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 DNA metabarcoding using nrITS2 provides highly qualitative and quantitative results for airborne pollen monitoring

2021 ◽  
pp. 150468
Author(s):  
Marcel Polling ◽  
Melati Sin ◽  
Letty A. de Weger ◽  
Arjen Speksnijder ◽  
Mieke J.F. Koenders ◽  
...  
Keyword(s):  
Airborne Pollen ◽  
Pollen Monitoring ◽  
Qualitative And Quantitative ◽  
Dna Metabarcoding ◽  
Quantitative Results

scholarly journals Detection of a new non-native freshwater species by DNA metabarcoding of environmental samples — first record of Gammarus fossarum in the UK

2017 ◽  
Vol 12 (2) ◽  
pp. 177-189 ◽  
Author(s):  
Rosetta Blackman ◽  
Drew Constable ◽  
Christoph Hahn ◽  
Andrew Sheard ◽  
Jessica Durkota ◽  
...  
Keyword(s):  
Environmental Samples ◽  
First Record ◽  
Freshwater Species ◽  
Gammarus Fossarum ◽  
Dna Metabarcoding ◽  
The Uk

scholarly journals Illumina iSeq 100 and MiSeq exhibit similar performance in freshwater fish environmental DNA metabarcoding

2020 ◽  
Author(s):  
Ryohei Nakao ◽  
Ryutei Inui ◽  
Yoshihisa Akamatsu ◽  
Masuji Goto ◽  
Hideyuki Doi ◽  
...  
Keyword(s):  
Environmental Samples ◽  
Environmental Dna ◽  
Illumina Miseq ◽  
Common Species ◽  
Species Numbers ◽  
Amplicon Library ◽  
Dna Metabarcoding ◽  
Biomonitoring Tool ◽  
The Common ◽  
Conventional Methods

AbstractEnvironmental DNA (eDNA) analysis is a method of detecting DNA from environmental samples, and it is used as a biomonitoring tool. In recent studies, Illumina MiSeq has been the most extensively used tool for eDNA metabarcoding, one of the eDNA analysis approaches. The Illumina iSeq 100 (hereafter, iSeq) is one of the numerous high-throughput sequencers (HTS). It has a relatively simple workflow and is potentially more affordable than other sequencers for deployment in HTS environments. However, to date, only a few studies have adopted iSeq, and its utility in eDNA metabarcoding has still not been investigated comprehensively. In the present study, we applied fish eDNA metabarcoding to river and lake environmental samples using iSeq and MiSeq approaches. We also assessed differences in fish species detectability among iSeq, MiSeq, and conventional approaches. Twenty-seven river and 13 lake samples were amplified using MiFish primers and sequenced with iSeq and MiSeq, respectively. The iSeq and MiSeq metabarcoding achieved high detectability for fish taxa in the ecosystems. Species numbers and compositions in each river detected using iSeq were almost consistent with those of MiSeq, indicating detectability of both techniques was comparable. The comparison of the species compositions of the two HTSs with those of conventional methods showed that the common species between each HTS and the conventional methods were exactly similar. According to the results, if the same amplicon library were used for sequencing, there would be negligible detectability differences between iSeq and MiSeq based on eDNA metabarcoding.

scholarly journals Use of Laser Microdissection for Phylogenetic Characterization of Polyphosphate-Accumulating Bacteria

2008 ◽  
Vol 74 (13) ◽  
pp. 4231-4235 ◽  
Author(s):  
Stefanie Gloess ◽  
Hans-Peter Grossart ◽  
Martin Allgaier ◽  
Stefan Ratering ◽  
Michael Hupfer
Keyword(s):  
Activated Sludge ◽  
Lake Sediment ◽  
Environmental Samples ◽  
Dna Analysis ◽  
Laser Microdissection ◽  
Taxonomic Identification ◽  
Phylogenetic Characterization ◽  
Uncultured Bacteria ◽  
Novel Approach

ABSTRACT Our novel approach for taxonomic identification of uncultured bacteria harboring specific physiological features in complex environmental samples combines cell collection by laser microdissection and subsequent DNA analysis. The newly developed approach was successfully tested for collection and phylogenetic characterization of polyphosphate-accumulating bacteria in activated sludge and lake sediment.

scholarly journals Propylene glycol-based antifreeze as an effective preservative for DNA metabarcoding of benthic arthropods

2020 ◽  
Author(s):  
Chloe V. Robinson ◽  
Teresita M. Porter ◽  
Michael T.G. Wright ◽  
Mehrdad Hajibabaei
Keyword(s):  
Propylene Glycol ◽  
Environmental Samples ◽  
Absolute Ethanol ◽  
Processing Methods ◽  
Suitable Alternative ◽  
Remote Areas ◽  
Dna Metabarcoding ◽  
Ethanol Evaporation ◽  
Type Site ◽  
Integrity Of Dna

AbstractMaintaining the integrity of DNA in bulk environmental samples from source to laboratory is crucial for capturing the true range of taxa present within an ecosystem. Preservation consideration of DNA is particularly important if samples are being collected in remote areas and by non-specialist ‘citizen scientists’ in nationwide programs. Traditionally, absolute ethanol is used as the preferred preservative for environmental samples collected for downstream DNA metabarcoding analyses. However, transport, shipping and DNA extraction of samples preserved in ethanol is a lengthy procedure due to safety restrictions and the requirement of full ethanol evaporation prior to extraction. We examined the efficacy of an easily accessible, non-toxic propylene glycol-based antifreeze as an alternative to absolute ethanol for preserving macroinvertebrate DNA from bulk-benthos DNA samples. We tested the differences in both cytochrome oxidase I (COI) exact sequence variants (ESVs) and COI taxonomic orders detected in both ethanol and antifreeze samples using two processing methods (no evaporation of preservative versus full evaporation). In addition, we assessed the detection of families and genera within the Arthropoda phylum for preservative type, site and processing method. Our results suggest that antifreeze is a suitable alternative to ethanol, a greater global ESV richness reported for antifreeze samples. Additionally, a higher proportion of arthropod reads in ESVs were detected in antifreeze (average 69%) compared with ethanol (average 53%). Finally, antifreeze samples produced similar results for the different processing methods, whereas ethanol samples failed to produce similar results without prior evaporation. Although ethanol is currently widely used for DNA preservation, our results demonstrate that by using antifreeze, it is possible to achieve similar taxonomic coverage and community assemblages of bulk-benthos DNA samples for macroinvertebrates, with the added simplicity and shorter laboratory processing time achieved using an easily available, unregulated preservative.

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.

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