scholarly journals Prospects and challenges of implementing DNA metabarcoding for high-throughput insect surveillance

GigaScience ◽  
2019 ◽  
Vol 8 (8) ◽  
Author(s):  
Alexander M Piper ◽  
Jana Batovska ◽  
Noel O I Cogan ◽  
John Weiss ◽  
John Paul Cunningham ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
Scale Up ◽  
Pest Species ◽  
Rapid Diagnostics ◽  
Invasive Insects ◽  
Sequencing Technologies ◽  
Dna Metabarcoding ◽  
Diagnostic Applications ◽  
Sequencing Platforms

AbstractTrap-based surveillance strategies are widely used for monitoring of invasive insect species, aiming to detect newly arrived exotic taxa as well as track the population levels of established or endemic pests. Where these surveillance traps have low specificity and capture non-target endemic species in excess of the target pests, the need for extensive specimen sorting and identification creates a major diagnostic bottleneck. While the recent development of standardized molecular diagnostics has partly alleviated this requirement, the single specimen per reaction nature of these methods does not readily scale to the sheer number of insects trapped in surveillance programmes. Consequently, target lists are often restricted to a few high-priority pests, allowing unanticipated species to avoid detection and potentially establish populations.DNA metabarcoding has recently emerged as a method for conducting simultaneous, multi-species identification of complex mixed communities and may lend itself ideally to rapid diagnostics of bulk insect trap samples. Moreover, the high-throughput nature of recent sequencing platforms could enable the multiplexing of hundreds of diverse trap samples on a single flow cell, thereby providing the means to dramatically scale up insect surveillance in terms of both the quantity of traps that can be processed concurrently and number of pest species that can be targeted. In this review of the metabarcoding literature, we explore how DNA metabarcoding could be tailored to the detection of invasive insects in a surveillance context and highlight the unique technical and regulatory challenges that must be considered when implementing high-throughput sequencing technologies into sensitive diagnostic applications.

scholarly journals Improved Selection of Internal Transcribed Spacer-Specific Primers Enables Quantitative, Ultra-High-Throughput Profiling of Fungal Communities

2013 ◽  
Vol 79 (8) ◽  
pp. 2519-2526 ◽  
Author(s):  
Nicholas A. Bokulich ◽  
David A. Mills
Keyword(s):  
High Throughput ◽  
Internal Transcribed Spacer ◽  
High Throughput Sequencing ◽  
Work Flow ◽  
Fungal Communities ◽  
Amplification Bias ◽  
Sequencing Technologies ◽  
Yeast Community ◽  
Primer Sets ◽  
Sequencing Platforms

ABSTRACTUltra-high-throughput sequencing (HTS) of fungal communities has been restricted by short read lengths and primer amplification bias, slowing the adoption of newer sequencing technologies to fungal community profiling. To address these issues, we evaluated the performance of several common internal transcribed spacer (ITS) primers and designed a novel primer set and work flow for simultaneous quantification and species-level interrogation of fungal consortia. Primer comparison and validation were predictedin silicoand by sequencing a “mock community” of mixed yeast species to explore the challenges of amplicon length and amplification bias for reconstructing defined yeast community structures. The amplicon size and distribution of this primer set are smaller than for all preexisting ITS primer sets, maximizing sequencing coverage of hypervariable ITS domains by very-short-amplicon, high-throughput sequencing platforms. This feature also enables the optional integration of quantitative PCR (qPCR) directly into the HTS preparatory work flow by substituting qPCR with these primers for standard PCR, yielding quantification of individual community members. The complete work flow described here, utilizing any of the qualified primer sets evaluated, can rapidly profile mixed fungal communities and capably reconstructed well-characterized beer and wine fermentation fungal communities.

scholarly journals Standards for Sequencing Viral Genomes in the Era of High-Throughput Sequencing

mBio ◽  
2014 ◽  
Vol 5 (3) ◽  
Author(s):  
Jason T. Ladner ◽  
Brett Beitzel ◽  
Patrick S. G. Chain ◽  
Matthew G. Davenport ◽  
Eric Donaldson ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
Cost Benefit ◽  
Genome Sequences ◽  
Common Component ◽  
Viral Genomes ◽  
Genome Finishing ◽  
Sequencing Technologies ◽  
Trade Offs ◽  
Sequencing Platforms

ABSTRACT Thanks to high-throughput sequencing technologies, genome sequencing has become a common component in nearly all aspects of viral research; thus, we are experiencing an explosion in both the number of available genome sequences and the number of institutions producing such data. However, there are currently no common standards used to convey the quality, and therefore utility, of these various genome sequences. Here, we propose five “standard” categories that encompass all stages of viral genome finishing, and we define them using simple criteria that are agnostic to the technology used for sequencing. We also provide genome finishing recommendations for various downstream applications, keeping in mind the cost-benefit trade-offs associated with different levels of finishing. Our goal is to define a common vocabulary that will allow comparison of genome quality across different research groups, sequencing platforms, and assembly techniques.

scholarly journals Assessing genotyping errors in mammalian museum study skins using high-throughput genotyping-by-sequencing

2021 ◽  
Author(s):  
Stella C. Yuan ◽  
Eric Malekos ◽  
Melissa T. R. Hawkins
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
Massively Parallel Sequencing ◽  
Massively Parallel ◽  
Museum Specimens ◽  
Museum Specimen ◽  
Genotyping Errors ◽  
Allelic Dropout ◽  
Parallel Sequencing ◽  
Sequencing Technologies

AbstractThe use of museum specimens held in natural history repositories for population and conservation genetic research is increasing in tandem with the use of massively parallel sequencing technologies. Short Tandem Repeats (STRs), or microsatellite loci, are commonly used genetic markers in wildlife and population genetic studies. However, they traditionally suffered from a host of issues including length homoplasy, high costs, low throughput, and difficulties in reproducibility across laboratories. Massively parallel sequencing technologies can address these problems, but the incorporation of museum specimen derived DNA suffers from significant fragmentation and exogenous DNA contamination. Combatting these issues requires extra measures of stringency in the lab and during data analysis, yet there have not been any high-throughput sequencing studies evaluating microsatellite allelic dropout from museum specimen extracted DNA. In this study, we evaluate genotyping errors derived from mammalian museum skin DNA extracts for previously characterized microsatellites across PCR replicates utilizing high-throughput sequencing. We found it useful to classify samples based on DNA concentration, which determined the rate by which genotypes were accurately recovered. Longer microsatellites performed worse in all museum specimens. Allelic dropout rates across loci were dependent on sample quantity, with high concentration museum specimens performing as well and recovering quality metrics nearly as high as the frozen tissue sample. Based on our results, we provide a set of best practices for quality assurance and incorporation of reliable genotypes from museum specimens.

scholarly journals Detection of novel allelic variations in soybean mutant population using Tilling by Sequencing

2019 ◽  
Author(s):  
Reneth Millas ◽  
Mary Espina ◽  
CM Sabbir Ahmed ◽  
Angelina Bernardini ◽  
Ekundayo Adeleke ◽  
...  
Keyword(s):  
Fatty Acid ◽  
High Throughput ◽  
Reverse Genetics ◽  
High Throughput Sequencing ◽  
Fatty Acid Biosynthesis ◽  
Induced Mutations ◽  
Mutant Population ◽  
Sequencing Technologies ◽  
Allelic Variations ◽  
Tilling By Sequencing

ABSTRACTOne of the most important tools in genetic improvement is mutagenesis, which is a useful tool to induce genetic and phenotypic variation for trait improvement and discovery of novel genes. JTN-5203 (MG V) mutant population was generated using an induced ethyl methane sulfonate (EMS) mutagenesis and was used for detection of induced mutations in FAD2-1A and FAD2-1B genes using reverse genetics approach. Optimum concentration of EMS was used to treat 15,000 bulk JTN-5203 seeds producing 1,820 M2 population. DNA was extracted, normalized, and pooled from these individuals. Specific primers were designed from FAD2-1A and FAD2-1B genes that are involved in the fatty acid biosynthesis pathway for further analysis using next-generation sequencing. High throughput mutation discovery through TILLING-by-Sequencing approach was used to detect novel allelic variations in this population. Several mutations and allelic variations with high impacts were detected for FAD2-1A and FAD2-1B. This includes GC to AT transition mutations in FAD2-1A (20%) and FAD2-1B (69%). Mutation density for this population is estimated to be about 1/136kb. Through mutagenesis and high-throughput sequencing technologies, novel alleles underlying the mutations observed in mutants with reduced polyunsaturated fatty acids will be identified, and these mutants can be further used in breeding soybean lines with improved fatty acid profile, thereby developing heart-healthy-soybeans.

scholarly journals Assessing pollution of aquatic environments with diatoms’ DNA metabarcoding: experience and developments from France water framework directive networks

2019 ◽  
Vol 3 ◽  
Author(s):  
Vasselon Valentin ◽  
Rimet Frédéric ◽  
Domaizon Isabelle ◽  
Monnier Olivier ◽  
Reyjol Yorick ◽  
...  
Keyword(s):  
Water Framework Directive ◽  
High Throughput Sequencing ◽  
Ecological Status ◽  
Aquatic Environments ◽  
Morphological Identification ◽  
The Past ◽  
Sequencing Technologies ◽  
Dna Metabarcoding ◽  
Morphological Approach ◽  
Status Assessment

Ecological status assessment of watercourses is based on the calculation of quality indices using pollution sensitivity of targeted biological groups, including diatoms. The determination and quantification of diatom species is generally based on microscopic morphological identification, which requires expertise and is time-consuming and costly. In Europe, this morphological approach is legally imposed by standards and regulatory decrees by the Water Framework Directive (WFD). Over the past decade, a DNA-based molecular biology approach has newly been developed to identify species based on genetic criteria rather than morphological ones (i.e. DNA metabarcoding). In combination with high throughput sequencing technologies, metabarcoding makes it possible both to identify all species present in an environmental sample and to process several hundred samples in parallel. This article presents the results of two recent studies carried out on the WFD networks of rivers of Mayotte (2013–2018) and metropolitan France (2016–2018). These studies aimed at testing the potential application of metabarcoding for biomonitoring in the context of the WFD. We discuss the various methodological developments and optimisations that have been made to make the taxonomic inventories of diatoms produced by metabarcoding more reliable, particularly in terms of species quantification. We present the results of the application of this DNA approach on more than 500 river sites, comparing them with those obtained using the standardised morphological method. Finally, we discuss the potential of metabarcoding for routine application, its limits of application and propose some recommendations for future implementation in WFD.

scholarly journals Adenosine-to-inosine RNA editing may be implicated in human pathogenesis

2019 ◽  
pp. 22-25
Author(s):  
AA Kliuchnikova ◽  
SA Moshkovskii
Keyword(s):  
Immune Responses ◽  
Rna Editing ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Common Mechanism ◽  
Adenosine Deaminases ◽  
Human Transcriptome ◽  
Sequencing Technologies

Adenosine-to-inosine (A-to-I) RNA editing is a common mechanism of post-transcriptional modification in many metazoans including vertebrates; the process is catalyzed by adenosine deaminases acting on RNA (ADARs). Using high-throughput sequencing technologies resulted in finding thousands of RNA editing sites throughout the human transcriptome however, their functions are still poorly understood. The aim of this brief review is to draw attention of clinicians and biomedical researchers to ADAR-mediated RNA editing phenomenon and its possible implication in development of neuropathologies, antiviral immune responses and cancer.

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 Technological advancements and their importance for nematode identification

SOIL ◽  
2016 ◽  
Vol 2 (2) ◽  
pp. 257-270 ◽  
Author(s):  
Mohammed Ahmed ◽  
Melanie Sapp ◽  
Thomas Prior ◽  
Gerrit Karssen ◽  
Matthew Alan Back
Keyword(s):  
High Throughput ◽  
Crop Production ◽  
Large Scale ◽  
High Throughput Sequencing ◽  
Biological Indicators ◽  
Rapid Identification ◽  
Community Studies ◽  
Terrestrial Environments ◽  
Traditional Taxonomy ◽  
Sequencing Platforms

Abstract. Nematodes represent a species-rich and morphologically diverse group of metazoans known to inhabit both aquatic and terrestrial environments. Their role as biological indicators and as key players in nutrient cycling has been well documented. Some plant-parasitic species are also known to cause significant losses to crop production. In spite of this, there still exists a huge gap in our knowledge of their diversity due to the enormity of time and expertise often involved in characterising species using phenotypic features. Molecular methodology provides useful means of complementing the limited number of reliable diagnostic characters available for morphology-based identification. We discuss herein some of the limitations of traditional taxonomy and how molecular methodologies, especially the use of high-throughput sequencing, have assisted in carrying out large-scale nematode community studies and characterisation of phytonematodes through rapid identification of multiple taxa. We also provide brief descriptions of some the current and almost-outdated high-throughput sequencing platforms and their applications in both plant nematology and soil ecology.

Sign in / Sign up

Export Citation Format

Share Document