Variation in “bank of microscopic forms” in urchin barren coast: detection using DNA metabarcoding based on high-throughput sequencing

2020 ◽  
Vol 32 (3) ◽  
pp. 2115-2124
Author(s):  
Shingo Akita ◽  
Hiroki Murasawa ◽  
Yoshihito Takano ◽  
Yoko Kawakami ◽  
Daisuke Fujita ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
Dna Metabarcoding ◽  
Urchin Barren

scholarly journals DNA-Based Herbal Teas’ Authentication: An ITS2 and psbA-trnH Multi-Marker DNA Metabarcoding Approach

Plants ◽  
2021 ◽  
Vol 10 (10) ◽  
pp. 2120
Author(s):  
Jessica Frigerio ◽  
Giulia Agostinetto ◽  
Valerio Mezzasalma ◽  
Fabrizio De De Mattia ◽  
Massimo Labra ◽  
...  
Keyword(s):  
Quality Control ◽  
Quantitative Analysis ◽  
Medicinal Plants ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
The Other ◽  
Plant Component ◽  
Identification Rate ◽  
Dna Metabarcoding ◽  
Therapeutic Properties

Medicinal plants have been widely used in traditional medicine due to their therapeutic properties. Although they are mostly used as herbal infusion and tincture, employment as ingredients of food supplements is increasing. However, fraud and adulteration are widespread issues. In our study, we aimed at evaluating DNA metabarcoding as a tool to identify product composition. In order to accomplish this, we analyzed fifteen commercial products with DNA metabarcoding, using two barcode regions: psbA-trnH and ITS2. Results showed that on average, 70% (44–100) of the declared ingredients have been identified. The ITS2 marker appears to identify more species (n = 60) than psbA-trnH (n = 35), with an ingredients’ identification rate of 52% versus 45%, respectively. Some species are identified only by one marker rather than the other. Additionally, in order to evaluate the quantitative ability of high-throughput sequencing (HTS) to compare the plant component to the corresponding assigned sequences, in the laboratory, we created six mock mixtures of plants starting both from biomass and gDNA. Our analysis also supports the application of DNA metabarcoding for a relative quantitative analysis. These results move towards the application of HTS analysis for studying the composition of herbal teas for medicinal plants’ traceability and quality control.

scholarly journals PLANiTS: a curated sequence reference dataset for plant ITS DNA metabarcoding

Database ◽  
2020 ◽  
Vol 2020 ◽  
Author(s):  
Elisa Banchi ◽  
Claudio G Ametrano ◽  
Samuele Greco ◽  
David Stanković ◽  
Lucia Muggia ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
Its Region ◽  
Computational Effort ◽  
Its Sequences ◽  
Reference Dataset ◽  
Bioinformatic Pipeline ◽  
Taxonomic Level ◽  
Dna Metabarcoding ◽  
Reference Databases

Abstract DNA metabarcoding combines DNA barcoding with high-throughput sequencing to identify different taxa within environmental communities. The ITS has already been proposed and widely used as universal barcode marker for plants, but a comprehensive, updated and accurate reference dataset of plant ITS sequences has not been available so far. Here, we constructed reference datasets of Viridiplantae ITS1, ITS2 and entire ITS sequences including both Chlorophyta and Streptophyta. The sequences were retrieved from NCBI, and the ITS region was extracted. The sequences underwent identity check to remove misidentified records and were clustered at 99% identity to reduce redundancy and computational effort. For this step, we developed a script called ‘better clustering for QIIME’ (bc4q) to ensure that the representative sequences are chosen according to the composition of the cluster at a different taxonomic level. The three datasets obtained with the bc4q script are PLANiTS1 (100 224 sequences), PLANiTS2 (96 771 sequences) and PLANiTS (97 550 sequences), and all are pre-formatted for QIIME, being this the most used bioinformatic pipeline for metabarcoding analysis. Being curated and updated reference databases, PLANiTS1, PLANiTS2 and PLANiTS are proposed as a reliable, pivotal first step for a general standardization of plant DNA metabarcoding studies. The bc4q script is presented as a new tool useful in each research dealing with sequences clustering. Database URL: https://github.com/apallavicini/bc4q; https://github.com/apallavicini/PLANiTS.

scholarly journals Sorting things out - assessing effects of unequal specimen biomass on DNA metabarcoding

2016 ◽  
Author(s):  
Vasco Elbrecht ◽  
Bianca Peinert ◽  
Florian Leese
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
Sequencing Depth ◽  
West Germany ◽  
Coi Gene ◽  
Mountain Stream ◽  
Detection Rates ◽  
Size Classes ◽  
Dna Metabarcoding ◽  
Size Sorting

1) Environmental bulk samples often contain many taxa with biomass differences of several orders of magnitude. This can be problematic in DNA metabarcoding and metagenomic high throughput sequencing approaches, as large specimens contribute over proportionally much DNA template. Thus a few specimens of high biomass will dominate the dataset, potentially leading to smaller specimens remaining undetected. Sorting of samples and balancing the amounts of tissue used per size fraction should improve detection rates, but has not been systematically tested. 2) Here we tested the effects of size sorting on taxa detection using freshwater macroinvertebrates. Kick sampling was performed at two locations of a low-mountain stream in West Germany, specimens were morphologically identified and sorted into small, medium and large size classes (< 2.5x5, 5x10 and up to 10x20 mm). Tissue from the 3 size categories was extracted individually, and pooled to simulate bulk samples that were not sorted and samples which were sorted and then pooled proportionately by specimen size. DNA from all 5 extractions of both samples was amplified using 4 different freshwater primer sets for the COI gene and sequenced on a HiSeq Illumina sequencer. 3) Sorting taxa by size and pooling them proportionately according to their abundance lead to a more equal amplification compared to the processing of complete samples without sorting. The sorted samples recovered 30% more taxa than the unsorted samples, at the same sequencing depth. Our results imply that sequencing depth can be decreased ~ 5 fold when sorting the samples into three size classes. 4) Our results demonstrate that even a coarse size sorting can substantially improve detection rates. While high throughput sequencing will become more accessible and cheaper within the next years, sorting bulk samples by specimen biomass is a simple yet efficient method to reduce current sequencing costs.

scholarly journals Scaling up DNA metabarcoding for freshwater macrozoobenthos monitoring

2018 ◽  
Author(s):  
Vasco Elbrecht ◽  
Dirk Steinke
Keyword(s):  
High Throughput ◽  
Illumina Sequencing ◽  
Large Scale ◽  
High Throughput Sequencing ◽  
Scaling Up ◽  
The Past ◽  
Dna Metabarcoding ◽  
Primer Sets ◽  
Fusion Primer ◽  
Positive Controls

The viability of DNA metabarcoding for assessment of freshwater macrozoobenthos has been demonstrated over the past years. It matured to a stage where it can be applied to monitoring at a large scale, keeping pace with increased high throughput sequencing (HTS) capacity. However, workflows and sample tagging need to be optimized to accommodate for hundreds of samples within a single sequencing run. We here conceptualize a streamlined metabarcoding workflow, in which samples are processed in 96-well plates. Each sample is replicated starting with tissue extraction. Negative and positive controls are included to ensure data reliability. With our newly developed fusion primer sets for the BF2+BR2 primer pair up to three 96-well plates (288 wells) can be uniquely tagged for a single Illumina sequencing run. By including Illumina indices tagging can be extended to thousands of samples. We hope that our metabarcoding workflow will be used as a practical guide for future large-scale biodiversity assessments involving freshwater invertebrates. However, we also want to point out that this is just one approach, and that we hope this article will stimulate discussion and publication of alternatives and extensions.

scholarly journals Scaling up DNA metabarcoding for freshwater macrozoobenthos monitoring

2018 ◽  
Author(s):  
Vasco Elbrecht ◽  
Dirk Steinke
Keyword(s):  
High Throughput ◽  
Illumina Sequencing ◽  
Large Scale ◽  
High Throughput Sequencing ◽  
Scaling Up ◽  
The Past ◽  
Dna Metabarcoding ◽  
Primer Sets ◽  
Fusion Primer ◽  
Positive Controls

The viability of DNA metabarcoding for assessment of freshwater macrozoobenthos has been demonstrated over the past years. It matured to a stage where it can be applied to monitoring at a large scale, keeping pace with increased high throughput sequencing (HTS) capacity. However, workflows and sample tagging need to be optimized to accommodate for hundreds of samples within a single sequencing run. We here conceptualize a streamlined metabarcoding workflow, in which samples are processed in 96-well plates. Each sample is replicated starting with tissue extraction. Negative and positive controls are included to ensure data reliability. With our newly developed fusion primer sets for the BF2+BR2 primer pair up to three 96-well plates (288 wells) can be uniquely tagged for a single Illumina sequencing run. By including Illumina indices tagging can be extended to thousands of samples. We hope that our metabarcoding workflow will be used as a practical guide for future large-scale biodiversity assessments involving freshwater invertebrates. However, we also want to point out that this is just one approach, and that we hope this article will stimulate discussion and publication of alternatives and extensions.

scholarly journals Scaling up DNA metabarcoding for freshwater macrozoobenthos monitoring

2018 ◽  
Author(s):  
Vasco Elbrecht ◽  
Dirk Steinke
Keyword(s):  
High Throughput ◽  
Illumina Sequencing ◽  
Large Scale ◽  
High Throughput Sequencing ◽  
Scaling Up ◽  
The Past ◽  
Dna Metabarcoding ◽  
Primer Sets ◽  
Fusion Primer ◽  
Positive Controls

The viability of DNA metabarcoding for assessment of freshwater macrozoobenthos has been demonstrated over the past years. It matured to a stage where it can be applied to monitoring at a large scale, keeping pace with increased high throughput sequencing (HTS) capacity. However, workflows and sample tagging need to be optimized to accommodate for hundreds of samples within a single sequencing run. We here conceptualize a streamlined metabarcoding workflow, in which samples are processed in 96-well plates. Each sample is replicated starting with tissue extraction. Negative and positive controls are included to ensure data reliability. With our newly developed fusion primer sets for the BF2+BR2 primer pair up to three 96-well plates (288 wells) can be uniquely tagged for a single Illumina sequencing run. By including Illumina indices, tagging can be extended to thousands of samples. We hope that our metabarcoding workflow will be used as a practical guide for future large-scale biodiversity assessments involving freshwater invertebrates. However, we also want to point out that this is just one possible metabarcoding approach, and that we hope this article will stimulate discussion and publication of alternatives and extensions.

scholarly journals Scaling up DNA metabarcoding for freshwater macrozoobenthos monitoring

2018 ◽  
Author(s):  
Vasco Elbrecht ◽  
Dirk Steinke
Keyword(s):  
High Throughput ◽  
Illumina Sequencing ◽  
Large Scale ◽  
High Throughput Sequencing ◽  
Scaling Up ◽  
The Past ◽  
Dna Metabarcoding ◽  
Primer Sets ◽  
Fusion Primer ◽  
Positive Controls

The viability of DNA metabarcoding for assessment of freshwater macrozoobenthos has been demonstrated over the past years. It matured to a stage where it can be applied to monitoring at a large scale, keeping pace with increased high throughput sequencing (HTS) capacity. However, workflows and sample tagging need to be optimized to accommodate for hundreds of samples within a single sequencing run. We here conceptualize a streamlined metabarcoding workflow, in which samples are processed in 96-well plates. Each sample is replicated starting with tissue extraction. Negative and positive controls are included to ensure data reliability. With our newly developed fusion primer sets for the BF2+BR2 primer pair up to three 96-well plates (288 wells) can be uniquely tagged for a single Illumina sequencing run. By including Illumina indices, tagging can be extended to thousands of samples. We hope that our metabarcoding workflow will be used as a practical guide for future large-scale biodiversity assessments involving freshwater invertebrates. However, we also want to point out that this is just one possible metabarcoding approach, and that we hope this article will stimulate discussion and publication of alternatives and extensions.

scholarly journals Scaling up DNA metabarcoding for freshwater macrozoobenthos monitoring

2018 ◽  
Author(s):  
Vasco Elbrecht ◽  
Dirk Steinke
Keyword(s):  
High Throughput ◽  
Illumina Sequencing ◽  
Large Scale ◽  
High Throughput Sequencing ◽  
Scaling Up ◽  
The Past ◽  
Dna Metabarcoding ◽  
Primer Sets ◽  
Fusion Primer ◽  
Positive Controls

The viability of DNA metabarcoding for assessment of freshwater macrozoobenthos has been demonstrated over the past years. It matured to a stage where it can be applied to monitoring at a large scale, keeping pace with increased high throughput sequencing (HTS) capacity. However, workflows and sample tagging need to be optimized to accommodate for hundreds of samples within a single sequencing run. We here conceptualize a streamlined metabarcoding workflow, in which samples are processed in 96-well plates. Each sample is replicated starting with tissue extraction. Negative and positive controls are included to ensure data reliability. With our newly developed fusion primer sets for the BF2+BR2 primer pair up to three 96-well plates (288 wells) can be uniquely tagged for a single Illumina sequencing run. By including Illumina indices, tagging can be extended to thousands of samples. We hope that our metabarcoding workflow will be used as a practical guide for future large-scale biodiversity assessments involving freshwater invertebrates. However, we also want to point out that this is just one possible metabarcoding approach, and that we hope this article will stimulate discussion and publication of alternatives and extensions.

scholarly journals How can integrated morphotaxonomy- and metabarcoding-based diatom assemblage analyses best contribute to the ecological assessment of streams?

2021 ◽  
Vol 5 ◽  
Author(s):  
Panayiota Pissaridou ◽  
Marco Cantonati ◽  
Agnes Bouchez ◽  
Iakovos Tziortzis ◽  
Gerald Dörflinger ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
Environmental Changes ◽  
Eastern Mediterranean ◽  
Ecological Status ◽  
Monitoring Network ◽  
Bioinformatic Analysis ◽  
Nutrient Concentrations ◽  
Mediterranean Country ◽  
Dna Metabarcoding

Environmental conditions, such as nutrient concentrations, salinity, elevation etc., shape diatom assemblages of periphytic biofilms. These assemblages respond rapidly to environmental changes, a fact which makes diatoms valuable bioindicators. Hence, freshwater biomonitoring programmes currently use diatom indices (e.g. EU Water Framework Directive - WFD). To date, microscopy-based assessments require high taxonomic expertise for diatom identification at the species level. High-throughput technologies now provide cost-effective identification approaches that are promising, complementary or alternative tools for bioassessment. The suitability of the metabarcoding method is evaluated for the first time in the Cyprus streams WFD monitoring network, an eastern Mediterranean country with many endemic species and results are compared to the results acquired from the morphotaxonomic analysis. Morphotaxonomic identification was conducted microscopically, using the most updated taxonomic concepts, literature and online resources. At the same time, DNA metabarcoding involved the use of the rbcL 312 bp barcode, high-throughput sequencing and bioinformatic analysis. The ecological status was calculated using the IPS Index. Results show a positive correlation between morpho-taxonomic and molecular IPS scores. Discrepancies between the two methodologies are related to the limitations of both techniques. This study confirmed that Fistulifera saprophila can have a crucial role in key differences observed, as it negatively influences IPS scores and microscopy methods frequently overlook it. Importantly, gaps in the DNA barcoding reference databases lead to a positive overestimation in IPS scores. Overall, we conclude that DNA metabarcoding offsets the morphotaxonomic methodology for the ecological quality assessment of freshwaters.

scholarly journals Sorting things out - assessing effects of unequal specimen biomass on DNA metabarcoding

2017 ◽  
Author(s):  
Vasco Elbrecht ◽  
Bianca Peinert ◽  
Florian Leese
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
Size Fraction ◽  
Sequencing Depth ◽  
Coi Gene ◽  
Mountain Stream ◽  
Detection Rates ◽  
Size Classes ◽  
Dna Metabarcoding ◽  
Size Sorting

Environmental bulk samples often contain many taxa that vary several orders of magnitude in biomass. This can be problematic in DNA metabarcoding and metagenomic high-throughput sequencing approaches, as large specimens contribute disproportionately high amounts of DNA template. Thus, a few specimens of high biomass will dominate the dataset, potentially leading to smaller specimens remaining undetected. Sorting of samples by specimen size and balancing the amounts of tissue used per size fraction should improve detection rates, but this approach has not been systematically tested. Here we explored the effects of size sorting on taxa detection using two freshwater macroinvertebrate monitoring samples, collected from a low-mountain stream in Germany. Specimens were morphologically identified and sorted into three size classes (body size < 2.5x5, 5x10 and up to 10x20 mm). Tissue from each size category was extracted individually, and pooled to simulate samples that were not sorted by biomass ("Unsorted"). Additionally, size fractions were pooled so that each specimen contributed approximately equal amounts of biomass ("Sorted"). Mock samples were amplified using four different DNA metabarcoding primer sets targeting the Cytochrome c oxidase I (COI) gene. Sorting taxa by size and pooling them proportionately according to their abundance lead to a more equal amplification of taxa compared to the processing of complete samples without sorting. The sorted samples recovered 30% more taxa than the unsorted samples, at the same sequencing depth. Our results imply that sequencing depth can be decreased approximately five-fold when sorting the samples into three size classes and pooling by specimen abundance. Our study demonstrates that even a coarse size sorting can substantially improve taxa detection using DNA metabarcoding. While high throughput sequencing will become more accessible and cheaper within the next years, sorting bulk samples by specimen biomass is a simple yet efficient method to reduce current sequencing costs.

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