scholarly journals In silico and empirical evaluation of twelve COI & 16S metabarcoding primer sets for insectivorous diet analyses

2019 ◽  
Author(s):  
Orianne Tournayre ◽  
Maxime Leuchtmann ◽  
Ondine Filippi-Codaccioni ◽  
Marine Trillat ◽  
Sylvain Piry ◽  
...  
Keyword(s):  
In Silico ◽  
Empirical Evaluation ◽  
Environmental Dna ◽  
Degraded Dna ◽  
Taxonomic Identification ◽  
Mock Community ◽  
Predator Detection ◽  
Primer Sets ◽  
Diet Analyses

AbstractThis last decade, environmental DNA metabarcoding approaches have been developed and improved to minimize biological and technical biases; some challenges, however, remain, as the design of primers. Here we have performed a comprehensive assessment of ten COI and two 16S primer sets. We have combined in silico, in vivo-mock community of 33 arthropod taxa from 16 orders and guano analyses to identify primer sets that should maximize arthropod detection and taxonomic identification, whilst identifying bat species and minimizing labour time and cost. We have focused on two insectivorous bat species living in mixed-colonies, the greater horseshoe bat (Rhinolophus ferrumequinum) and Geoffroy’s bat (Myotis emarginatus). We have found that the level of primer degeneracy is the main factor influencing arthropod detection for in silico and mock community analyses, while the amplicon length is critical for the detection of arthropods from degraded DNA samples. Our results confirm the importance of performing predator detection and taxonomic identification, simultaneously with arthropod sequencing, as faeces samples can be contaminated by different insectivorous species. Moreover, amplifying bat DNA does not affect the primers’ capacity to detect arthropods. We therefore recommend the systematic simultaneous identification of predator and prey. Finally, we evidenced that one third of the prey occurrences are unreliable and probably not of primary interest in diet studies, which might decrease the relevance of combining several primer sets instead of using one efficient primer set. In conclusion, this study provides general criteria enabling the selection of primers whilst considering different scientific and methodological constraints.

scholarly journals In silico and empirical evaluation of twelve metabarcoding primer sets for insectivorous diet analyses

2020 ◽  
Vol 10 (13) ◽  
pp. 6310-6332 ◽  
Author(s):  
Orianne Tournayre ◽  
Maxime Leuchtmann ◽  
Ondine Filippi‐Codaccioni ◽  
Marine Trillat ◽  
Sylvain Piry ◽  
...  
Keyword(s):  
In Silico ◽  
Empirical Evaluation ◽  
Primer Sets ◽  
Diet Analyses

scholarly journals Short COI markers for freshwater macroinvertebrate metabarcoding

2017 ◽  
Author(s):  
Ecaterina Edith Vamos ◽  
Vasco Elbrecht ◽  
Florian Leese
Keyword(s):  
In Silico ◽  
Detection Efficiency ◽  
In Silico Analysis ◽  
Environmental Dna ◽  
Degraded Dna ◽  
Macroinvertebrate Communities ◽  
Universal Primer ◽  
Primer Sets ◽  
Mock Communities

Species diversity of metazoan bulk samples can be rapidly assessed using cytochrome c oxidase I (COI) metabarcoding. However, in some applications often only degraded DNA is available, e.g. from poorly conserved museum specimens, environmental DNA (eDNA) filtered from water or gut content analyses. Here universal primer sets targeting only a short COI fragment are advantageous, as they often can still amplify short DNA fragments. Using PrimerMiner, we optimised two universal primer sets targeting freshwater macroinvertebrates based on NCBI and BOLD reference sequences. The fwh1 and fwh2 primer sets targeting a 178 and 205 bp region were tested in vitro by sequencing previously used freshwater macroinvertebrate mock communities as well as three monitoring samples from Romanian streams of unknown composition. They were further evaluated in silico for their suitability to amplify other insect groups. The fwh1 primer sets showed the most consistent amplification in silico and in vitro , detecting 92% of the taxa present in the mock communities, and allowing clear differentiation between the three macroinvertebrate communities from the Romanian streams. In silico analysis indicates that the short primers are likely to perform well even for non-freshwater insects. Comparing the performance of the new fwh1 primer sets to a highly degenerate primer set targeting a longer fragment (BF2+BR2) revealed that detection efficiency is slightly lower for the new primer set. Nevertheless, the shorter new primer pairs might be useful for studies that have to rely on degraded or poorly conserved DNA and thus be of importance for biomonitoring, conservation biological or molecular ecological studies. Furthermore, our study highlights the need for in silico evaluation of primer sets in order to detect design errors in primers (fwhR2) and find optimal universal primer sets for the target taxa of interest.

scholarly journals Short COI markers for freshwater macroinvertebrate metabarcoding

2017 ◽  
Author(s):  
Ecaterina Edith Vamos ◽  
Vasco Elbrecht ◽  
Florian Leese
Keyword(s):  
In Silico ◽  
In Silico Analysis ◽  
Degraded Dna ◽  
Macroinvertebrate Communities ◽  
Universal Primer ◽  
Primer Sets ◽  
Clear Differentiation ◽  
Silico Analysis ◽  
Mock Communities

Species diversity of metazoan bulk samples can be rapidly assessed using Cytochrome c oxidase I (COI) metabarcoding. However, in cases where only degraded DNA is available, e.g. from poorly conserved museum specimens, eDNA filtered from water or gut content analyses, universal primer sets that amplify only a short COI fragment are advantageous. Using PrimerMiner, we optimised two universal primer sets targeting freshwater macroinvertebrates based on NCBI and BOLD reference sequences. The fwh1 and fwh2 primer sets targeting a 178 and 205 bp region were tested in vivo by sequencing previously used freshwater macroinvertebrate mock communities of known composition and three monitoring samples from Romanian streams. They were further evaluated in silico for their suitability to amplify other insect groups. The fwh1 primer sets showed the most consistent amplification in silico and in vivo , detecting 92% of the taxa present in the mock communities, and allowing clear differentiation between the three macroinvertebrate communities from the Romanian streams. In silico analysis indicates that the short primers are likely to perform well even for non-freshwater insects. Comparing the performance of the new fwh1 primer sets to a highly degenerate primer set targeting a longer fragment (BF2/BR2) revealed that efficiency is slightly lower for the new primer set. Nevertheless, the shorter new primer pairs might be useful for studies that have to rely on degraded or poorly conserved DNA and thus be of importance for biomonitoring, conservation biological or molecular ecological studies. Furthermore, our study highlights the need for in silico evaluation of primer sets in order to detect design errors in primers (fwhR2) and find optimal universal primer sets for the target taxa of interest.

scholarly journals Short COI markers for freshwater macroinvertebrate metabarcoding

2017 ◽  
Author(s):  
Ecaterina Edith Vamos ◽  
Vasco Elbrecht ◽  
Florian Leese
Keyword(s):  
In Silico ◽  
Detection Efficiency ◽  
In Silico Analysis ◽  
Environmental Dna ◽  
Degraded Dna ◽  
Macroinvertebrate Communities ◽  
Universal Primer ◽  
Primer Sets ◽  
Mock Communities

Species diversity of metazoan bulk samples can be rapidly assessed using cytochrome c oxidase I (COI) metabarcoding. However, in some applications often only degraded DNA is available, e.g. from poorly conserved museum specimens, environmental DNA (eDNA) filtered from water or gut content analyses. Here universal primer sets targeting only a short COI fragment are advantageous, as they often can still amplify short DNA fragments. Using PrimerMiner, we optimised two universal primer sets targeting freshwater macroinvertebrates based on NCBI and BOLD reference sequences. The fwh1 and fwh2 primer sets targeting a 178 and 205 bp region were tested in vitro by sequencing previously used freshwater macroinvertebrate mock communities as well as three monitoring samples from Romanian streams of unknown composition. They were further evaluated in silico for their suitability to amplify other insect groups. The fwh1 primer sets showed the most consistent amplification in silico and in vitro , detecting 92% of the taxa present in the mock communities, and allowing clear differentiation between the three macroinvertebrate communities from the Romanian streams. In silico analysis indicates that the short primers are likely to perform well even for non-freshwater insects. Comparing the performance of the new fwh1 primer sets to a highly degenerate primer set targeting a longer fragment (BF2+BR2) revealed that detection efficiency is slightly lower for the new primer set. Nevertheless, the shorter new primer pairs might be useful for studies that have to rely on degraded or poorly conserved DNA and thus be of importance for biomonitoring, conservation biological or molecular ecological studies. Furthermore, our study highlights the need for in silico evaluation of primer sets in order to detect design errors in primers (fwhR2) and find optimal universal primer sets for the target taxa of interest.

scholarly journals Validation of COI metabarcoding primers for terrestrial arthropods

PeerJ ◽  
2019 ◽  
Vol 7 ◽  
pp. e7745 ◽  
Author(s):  
Vasco Elbrecht ◽  
Thomas W.A. Braukmann ◽  
Natalia V. Ivanova ◽  
Sean W.J. Prosser ◽  
Mehrdad Hajibabaei ◽  
...  
Keyword(s):  
Degraded Dna ◽  
Taxonomic Resolution ◽  
Effect Of Temperature ◽  
Gut Contents ◽  
Minor Effect ◽  
Malaise Trap ◽  
Mock Community ◽  
Terrestrial Arthropods ◽  
Primer Sets ◽  
Trap Sample

Metabarcoding can rapidly determine the species composition of bulk samples and thus aids biodiversity and ecosystem assessment. However, it is essential to use primer sets that minimize amplification bias among taxa to maximize species recovery. Despite this fact, the performance of primer sets employed for metabarcoding terrestrial arthropods has not been sufficiently evaluated. This study tests the performance of 36 primer sets on a mock community containing 374 insect species. Amplification success was assessed with gradient PCRs and the 21 most promising primer sets selected for metabarcoding. These 21 primer sets were also tested by metabarcoding a Malaise trap sample. We identified eight primer sets, mainly those including inosine and/or high degeneracy, that recovered more than 95% of the species in the mock community. Results from the Malaise trap sample were congruent with the mock community, but primer sets generating short amplicons produced potential false positives. Taxon recovery from both mock community and Malaise trap sample metabarcoding were used to select four primer sets for additional evaluation at different annealing temperatures (40–60 °C) using the mock community. The effect of temperature varied by primer pair but overall it only had a minor effect on taxon recovery. This study reveals the weak performance of some primer sets employed in past studies. It also demonstrates that certain primer sets can recover most taxa in a diverse species assemblage. Thus, based our experimental set up, there is no need to employ several primer sets targeting the same gene region. We identify several suitable primer sets for arthropod metabarcoding, and specifically recommend BF3 + BR2, as it is not affected by primer slippage and provides maximal taxonomic resolution. The fwhF2 + fwhR2n primer set amplifies a shorter fragment and is therefore ideal when targeting degraded DNA (e.g., from gut contents).

scholarly journals Development and validation of DNA metabarcoding COI primers for aquatic invertebrates using the R package "PrimerMiner"

2016 ◽  
Author(s):  
Vasco Elbrecht ◽  
Florian Leese
Keyword(s):  
In Silico ◽  
Sequence Data ◽  
Dna Barcode ◽  
R Package ◽  
Gene Marker ◽  
Amplification Efficiency ◽  
Mock Community ◽  
Dna Metabarcoding ◽  
Primer Sets ◽  
High Base

1) DNA metabarcoding is a powerful tool to assess biodiversity by amplifying and sequencing a standardized gene marker region. However, typically used barcoding genes, such as the cytochrome c oxidase subunit I (COI) region for animals, are highly variable. Thus, different taxa in communities under study are often not amplified equally well and some might even remain undetected due to primer bias. To reduce these problems, optimized region- and/or ecosystem- specific metabarcoding primers are necessary. 2) We developed the R package PrimerMiner, which batch downloads DNA barcode gene sequences from BOLD and NCBI databases for specified target taxa and then applies sequence clustering to reduce biases introduced by differed number of available sequences per species. To design primers targeted for freshwater invertebrates, we downloaded COI data for the 15 most important invertebrate groups relevant for stream ecosystem assessment. Four primer sets with high base degeneracy were developed and their performance tested by sequencing ten mock community samples consisting each of 52 freshwater invertebrate taxa. Additionally, we evaluated the developed primers against other metabarcoding primers in silico using PrimerMiner. 3) Amplification and sequencing was successful for all ten mock community samples with the four different primer combinations. The developed primers varied in amplification efficiency and amount of taxa detected, but all primer sets detected more taxa than standard Folmer barcoding primers. Additionally, the BF / BR primers amplified taxa very consistently, the BF2+BR2 and BF2+BR1 primer combination even better than a previously tested ribosomal marker (16S). Except for the BF1+BR1 primer combination, all BF / BR primers detected all 42 insect taxa present in the mock samples. In silico evaluation of the developed primers showed that they are also likely to work very well on other non aquatic invertebrate samples. 4) With PrimerMiner, we here provide a useful tool to obtain relevant sequence data for targeted primer development and evaluation. Our sequence datasets generated with the newly developed metabarcoding primers demonstrate that the design of optimized primers with high base degeneracy is superior to classical markers and enable us to detect almost 100% of animal taxa present in a sample using the standard COI barcoding gene. Therefore, the PrimerMiner package and primers developed using this tool are useful beyond assessment of biodiversity in aquatic ecosystems.

Mechanisms of action of antihyperglycemic mexicanolides isolated from Swietenia humillis: in vivo and in silico approaches

Planta Medica ◽  
2016 ◽  
Vol 81 (S 01) ◽  
pp. S1-S381 ◽  
Author(s):  
B Ovalle-Magallanes ◽  
A Madariaga-Mazón ◽  
A Navarrete ◽  
R Mata
Keyword(s):  
In Silico ◽  
Mechanisms Of Action

Ruthenium Complexes for 1- and 2-Photon Photodynamic Therapy: From In Silico Prediction to In Vivo Applications

2020 ◽  
Author(s):  
Johannes Karges ◽  
Shi Kuang ◽  
Federica Maschietto ◽  
Olivier Blacque ◽  
Ilaria Ciofini ◽  
...  
Keyword(s):  
Photodynamic Therapy ◽  
In Silico ◽  
Aqueous Solubility ◽  
The Body ◽  
Photon Absorption ◽  
Multicellular Tumor Spheroids ◽  
Spectral Window ◽  
Photon Excitation ◽  
Polypyridine Complexes

<div>The use of photodynamic therapy (PDT) against cancer has received increasing attention overthe recent years. However, the application of the currently approved photosensitizers (PSs) is somehow limited by their poor aqueous solubility, aggregation, photobleaching and slow clearance from the body. To overcome these limitations, there is a need for the development of new classes of PSs with ruthenium(II) polypyridine complexes currently gaining momentum. However, these compounds generally lack significant absorption in the biological spectral window, limiting their application to treat deep-seated or large tumors. To overcome this drawback, ruthenium(II) polypyridine complexes designed in silico with (E,E’)-4,4´-bisstyryl 2,2´-bipyridine ligands showed impressive 1- and 2-Photon absorption up to a magnitude higher than the ones published so far. While non-toxic in the dark, these compounds were found phototoxic in various 2D monolayer cells, 3D multicellular tumor spheroids and be able to eradicate a multiresistant tumor inside a mouse model upon clinically relevant 1-Photon and 2 Photon excitation.</div>

Recent In Vitro and In Silico Advances in the Understanding of Intranasal Drug Delivery

2020 ◽  
Vol 26 ◽  
Author(s):  
John Chen ◽  
Andrew Martin ◽  
Warren H. Finlay
Keyword(s):  
Drug Delivery ◽  
In Silico ◽  
Local Drug Delivery ◽  
In Vivo Studies ◽  
Intranasal Drug Delivery ◽  
Nasal Sprays ◽  
In Silico Studies ◽  
Drug Delivery Devices

Background: Many drugs are delivered intranasally for local or systemic effect, typically in the form of droplets or aerosols. Because of the high cost of in vivo studies, drug developers and researchers often turn to in vitro or in silico testing when first evaluating the behavior and properties of intranasal drug delivery devices and formulations. Recent advances in manufacturing and computer technologies have allowed for increasingly realistic and sophisticated in vitro and in silico reconstructions of the human nasal airways. Objective: To perform a summary of advances in understanding of intranasal drug delivery based on recent in vitro and in silico studies. Conclusion: The turbinates are a common target for local drug delivery applications, and while nasal sprays are able to reach this region, there is currently no broad consensus across the in vitro and in silico literature concerning optimal parameters for device design, formulation properties and patient technique which would maximize turbinate deposition. Nebulizers are able to more easily target the turbinates, but come with the disadvantage of significant lung deposition. Targeting of the olfactory region of the nasal cavity has been explored for potential treatment of central nervous system conditions. Conventional intranasal devices, such as nasal sprays and nebulizers, deliver very little dose to the olfactory region. Recent progress in our understanding of intranasal delivery will be useful in the development of the next generation of intranasal drug delivery devices.

In-silico Studies of Isolated Phytoalkaloid Against Lipoxygenase: Study Based on Possible Correlation

2018 ◽  
Vol 21 (3) ◽  
pp. 215-221
Author(s):  
Haroon Khan ◽  
Muhammad Zafar ◽  
Helena Den-Haan ◽  
Horacio Perez-Sanchez ◽  
Mohammad Amjad Kamal
Keyword(s):  
In Silico ◽  
Docking Studies ◽  
In Vivo Studies ◽  
In Vitro Assays ◽  
Chronic Obstructive ◽  
Lipoxygenase Inhibitors ◽  
Lipoxygenase Inhibition ◽  
In Silico Studies

Aim and Objective: Lipoxygenase (LOX) enzymes play an important role in the pathophysiology of several inflammatory and allergic diseases including bronchial asthma, allergic rhinitis, atopic dermatitis, allergic conjunctivitis, rheumatoid arthritis and chronic obstructive pulmonary disease. Inhibitors of the LOX are believed to be an ideal approach in the treatment of diseases caused by its over-expression. In this regard, several synthetic and natural agents are under investigation worldwide. Alkaloids are the most thoroughly investigated class of natural compounds with outstanding past in clinically useful drugs. In this article, we have discussed various alkaloids of plant origin that have already shown lipoxygenase inhibition in-vitro with possible correlation in in silico studies. Materials and Methods: Molecular docking studies were performed using MOE (Molecular Operating Environment) software. Among the ten reported LOX alkaloids inhibitors, derived from plant, compounds 4, 2, 3 and 1 showed excellent docking scores and receptor sensitivity. Result and Conclusion: These compounds already exhibited in vitro lipoxygenase inhibition and the MOE results strongly correlated with the experimental results. On the basis of these in vitro assays and computer aided results, we suggest that these compounds need further detail in vivo studies and clinical trial for the discovery of new more effective and safe lipoxygenase inhibitors. In conclusion, these results might be useful in the design of new and potential lipoxygenase (LOX) inhibitors.

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