Mosquito Identification From Bulk Samples Using DNA Metabarcoding: a Protocol to Support Mosquito-Borne Disease Surveillance in Canada

2021 ◽  
Author(s):  
S Mechai ◽  
G Bilodeau ◽  
O Lung ◽  
M Roy ◽  
R Steeves ◽  
...  
Keyword(s):  
Disease Surveillance ◽  
High Throughput Sequencing ◽  
Light Trap ◽  
Illumina Miseq ◽  
Bioinformatics Pipeline ◽  
Eastern Canada ◽  
Cdc Light Trap ◽  
Dna Metabarcoding ◽  
Significant Public Health ◽  
A Site

Abstract Approximately 80 species of mosquitoes (Diptera: Culicidae) have been documented in Canada. Exotic species such as Aedes albopictus (Skuse) (Diptera: Culicidae) are becoming established. Recently occurring endemic mosquito-borne diseases (MBD) in Canada including West-Nile virus (WNV) and Eastern Equine Encephalitis (EEE) are having significant public health impacts. Here we explore the use of DNA metabarcoding to identify mosquitoes from CDC light-trap collections from two locations in eastern Canada. Two primer pairs (BF2-BR2 and F230) were used to amplify regions of the cytochrome c oxidase subunit I (CO1) gene. High throughput sequencing was conducted using an Illumina MiSeq platform and GenBank-based species identification was applied using a QIIME 1.9 bioinformatics pipeline. From a site in southeastern Ontario, Canada, 26 CDC light trap collections of 72 to >300 individual mosquitoes were used to explore the capacity of DNA metabarcoding to identify and quantify captured mosquitoes. The DNA metabarcoding method identified 33 species overall while 24 species were identified by key. Using replicates from each trap, the dried biomass needed to identify the majority of species was determined to be 76 mg (equivalent to approximately 72 mosquitoes), and at least two replicates from the dried biomass would be needed to reliably detect the majority of species in collections of 144–215 mosquitoes and three replicates would be advised for collections with >215 mosquitoes. This study supports the use of DNA metabarcoding as a mosquito surveillance tool in Canada which can help identify the emergence of new mosquito-borne disease potential threats.

scholarly journals Addressing the "Paradox of the Plankton": Using Metabarcoding to Explore Zooplankton Diversity Patterns Across Chemical Conditions in the Salish Sea

2021 ◽  
Vol 4 ◽  
Author(s):  
Haila Schultz ◽  
Carol Stepien ◽  
Julie Keister ◽  
Emily Norton ◽  
Sean McAllister ◽  
...  
Keyword(s):  
High Throughput Sequencing ◽  
Environmental Chemistry ◽  
Puget Sound ◽  
Illumina Miseq ◽  
Morphological Characters ◽  
Salish Sea ◽  
Bioinformatics Pipeline ◽  
Mitochondrial Coi ◽  
Chemical Conditions ◽  
Diversity Of Life

Traditional taxonomic analysis of zooplankton is time consuming, expensive, and unable to resolve the true species diversity of a community due to a lack of diagnostic morphological characters for many taxa. This is especially true for early life stages, undescribed, and cryptic species. This limitation has led to a dramatic under-estimation of the incredible diversity of life that inhabits the ocean, hindering our understanding of the environmental conditions that structure communities. Multi-gene metabarcode high-throughput sequencing (HTS) analyses entailing field sampling and bioinformatics offer new means to rapidly and accurately characterize the species identities, diversity, and composition of entire communities. We use multiple diagnostic Illumina MiSeq HTS metabarcode assays (for mitochondrial COI, 16SRNA, and 12SRNA gene regions) and a custom bioinformatics pipeline to analyze communities of invertebrates and fishes from zooplankton net tows collected concurrently with environmental chemistry data across Puget Sound in the southern Salish Sea. We compare results among sites for spring and autumn seasons, as a prelude to multi-year analyses. Findings show considerable divergence in species composition and diversity among sites and season, reflecting differences in salinity, pH, and proximity to the ocean. This approach has great potential for wide-spread use in monitoring programs to assess the diversity of marine plankton communities in conjunction with changing conditions, including ocean acidification, hypoxia, and global temperature rise.

scholarly journals DNA metabarcoding Uncover the Diet of Subterranean Rodents in China

2021 ◽  
Author(s):  
Xuxin Zhang ◽  
Yao Zou ◽  
Xiaoning Nan ◽  
Chongxuan Han
Keyword(s):  
High Throughput Sequencing ◽  
Dietary Habits ◽  
Alpha Diversity ◽  
Northern China ◽  
Illumina Miseq ◽  
Subterranean Rodents ◽  
Clustering Method ◽  
Mountain Area ◽  
Dna Metabarcoding ◽  
Gastric Contents

AbstractObjectiveIn the past, the zokor, which lived in northern China, caused great harm to agriculture and forestry production due to its large and sophisticated diet. Since the rat lives underground for most of its life, researchers know little about its dietary habits. Further understanding of its diet in the field is of important meaning for developing green and sustainable control strategies for the rat.MethodsLongde County in Liupan Mountain area of Ningxia Hui Autonomous Region was selected as the interest area to capture zokor and investigate the species of habitat plants.We selected chloroplast trnL gene and eukaryotic internal transcription spacer 1 (ITS 1) primers to amplify DNA from the gastric contents of zokor,and then sequenced on Illumina Miseq PE300 platform.ResultsThe gastric contents of Eospalax smithii (n=16) and E.cansus(n=9) were analyzed by operational taxonomic units (OTU) clustering and amplicon sequence variants(ASVs).The OTU clustering method obtained 2,995 OTUs, and the ASV method obtained 4,657 ASVs. The ASV method was better than the OTU clustering method, and the ASV method was adopted in the subsequent analysis. The food list of 32 families, 80 genera and 154 species was obtained by ASV method after the error was removed. The food composition of zokor was evaluated by relative abundance(%RA) method and frequency of occurrence(%ROO) method. At the Family level, it was found that zokor mainly fed on Asteraceae, Poaceae, Rosaceae, Pinaceae, Brassicaceae, Apiaceae, etc. At the Genus level, they are mainlyEchinops, Littledalea,Artemisia,Picea, Cirsium, Elymus and so on. The diet alpha diversity of E.cansus was slightly higher than that of E.smithii (P > 0.05). The correlation coefficient between Sobs index of alpha diversity and body weight of zokor was −0.382 (P = 0.059). The diet beta diversity proved that most zokors (22/25) clustered together, with low heterogeneity. They fed positively on Calamagrostis, Cirsium, Echinops, Medicago, Sanguisorba and Taraxacum. We found that zokor mainly fed on the roots of perennial herbs(PH), which were rich in water, carbohydrate, fat and protein, which provided an important energy source for its survival.ConclusionHigh-throughput sequencing(HTS) based DNA metabarcoding technology has well revealed the diet of zokor, which are generalist.

scholarly journals The Fecal Bacterial Microbiota in Horses with Equine Recurrent Uveitis

Animals ◽  
2021 ◽  
Vol 11 (3) ◽  
pp. 745
Author(s):  
Michelle Martin de Bustamante ◽  
Diego Gomez ◽  
Jennifer MacNicol ◽  
Ralph Hamor ◽  
Caryn Plummer
Keyword(s):  
Beta Diversity ◽  
High Throughput Sequencing ◽  
Illumina Miseq ◽  
Diversity Indices ◽  
Rrna Gene ◽  
Linear Discriminant ◽  
Alpha And Beta Diversity ◽  
Bacterial Microbiota ◽  
Healthy Control ◽  
Equine Recurrent Uveitis

The objective of this study was to describe and compare the fecal bacterial microbiota of horses with equine recurrent uveitis (ERU) and healthy horses using next-generation sequencing techniques. Fecal samples were collected from 15 client-owned horses previously diagnosed with ERU on complete ophthalmic examination. For each fecal sample obtained from a horse with ERU, a sample was collected from an environmentally matched healthy control with no evidence of ocular disease. The Illumina MiSeq sequencer was used for high-throughput sequencing of the V4 region of the 16S rRNA gene. The relative abundance of predominant taxa, and alpha and beta diversity indices were calculated and compared between groups. The phyla Firmicutes, Bacteroidetes, Verrucomicrobia, and Proteobacteria predominated in both ERU and control horses, accounting for greater than 60% of sequences. Based on linear discriminant analysis effect size (LEfSe), no taxa were found to be enriched in either group. No significant differences were observed in alpha and beta diversity indices between groups (p > 0.05 for all tests). Equine recurrent uveitis is not associated with alteration of the gastrointestinal bacterial microbiota when compared with healthy controls.

Population-specific genome graphs improve high-throughput sequencing data analysis: A case study on the Pan-African genome

2021 ◽  
Author(s):  
H. Serhat Tetikol ◽  
Kubra Narci ◽  
Deniz Turgut ◽  
Gungor Budak ◽  
Ozem Kalay ◽  
...  
Keyword(s):  
High Throughput Sequencing ◽  
Information Overload ◽  
African Ancestry ◽  
Sample Selection ◽  
Variant Calling ◽  
Population Diversity ◽  
Human Populations ◽  
Sequencing Data ◽  
Bioinformatics Pipeline ◽  
Graph Augmentation

ABSTRACTGraph-based genome reference representations have seen significant development, motivated by the inadequacy of the current human genome reference for capturing the diverse genetic information from different human populations and its inability to maintain the same level of accuracy for non-European ancestries. While there have been many efforts to develop computationally efficient graph-based bioinformatics toolkits, how to curate genomic variants and subsequently construct genome graphs remains an understudied problem that inevitably determines the effectiveness of the end-to-end bioinformatics pipeline. In this study, we discuss major obstacles encountered during graph construction and propose methods for sample selection based on population diversity, graph augmentation with structural variants and resolution of graph reference ambiguity caused by information overload. Moreover, we present the case for iteratively augmenting tailored genome graphs for targeted populations and test the proposed approach on the whole-genome samples of African ancestry. Our results show that, as more representative alternatives to linear or generic graph references, population-specific graphs can achieve significantly lower read mapping errors, increased variant calling sensitivity and provide the improvements of joint variant calling without the need of computationally intensive post-processing steps.

scholarly journals MiDRMpol: A High-Throughput Multiplexed Amplicon Sequencing Workflow to Quantify HIV-1 Drug Resistance Mutations against Protease, Reverse Transcriptase, and Integrase Inhibitors

Viruses ◽  
2019 ◽  
Vol 11 (9) ◽  
pp. 806
Author(s):  
Shambhu G. Aralaguppe ◽  
Anoop T. Ambikan ◽  
Manickam Ashokkumar ◽  
Milner M. Kumar ◽  
Luke Elizabeth Hanna ◽  
...  
Keyword(s):  
Drug Resistance ◽  
High Throughput ◽  
Large Scale ◽  
High Throughput Sequencing ◽  
Polymorphism Analysis ◽  
Resistance Mutations ◽  
Subtype C ◽  
Bioinformatics Pipeline ◽  
Drug Resistance Mutations ◽  
Hiv 1

The detection of drug resistance mutations (DRMs) in minor viral populations is of potential clinical importance. However, sophisticated computational infrastructure and competence for analysis of high-throughput sequencing (HTS) data lack at most diagnostic laboratories. Thus, we have proposed a new pipeline, MiDRMpol, to quantify DRM from the HIV-1 pol region. The gag-vpu region of 87 plasma samples from HIV-infected individuals from three cohorts was amplified and sequenced by Illumina HiSeq2500. The sequence reads were adapter-trimmed, followed by analysis using in-house scripts. Samples from Swedish and Ethiopian cohorts were also sequenced by Sanger sequencing. The pipeline was validated against the online tool PASeq (Polymorphism Analysis by Sequencing). Based on an error rate of <1%, a value of >1% was set as reliable to consider a minor variant. Both pipelines detected the mutations in the dominant viral populations, while discrepancies were observed in minor viral populations. In five HIV-1 subtype C samples, minor mutations were detected at the <5% level by MiDRMpol but not by PASeq. MiDRMpol is a computationally as well as labor efficient bioinformatics pipeline for the detection of DRM from HTS data. It identifies minor viral populations (<20%) of DRMs. Our method can be incorporated into large-scale surveillance of HIV-1 DRM.

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 The use of eDNA and DNA metabarcoding in monitoring the ecological condition of Norwegian lakes

2021 ◽  
Vol 4 ◽  
Author(s):  
Sara Atienza Casas ◽  
Markus Majaneva ◽  
Thomas Jensen ◽  
Marie Davey ◽  
Frode Fossøy ◽  
...  
Keyword(s):  
High Throughput Sequencing ◽  
Ecological Status ◽  
Ecological Condition ◽  
Molecular Techniques ◽  
Taxonomic Resolution ◽  
Potential Cost ◽  
Running Water ◽  
National Monitoring ◽  
Monitoring Programs ◽  
Dna Metabarcoding

Biodiversity assessments using molecular identification of organisms through high-throughput sequencing techniques have been a game changer in ecosystem monitoring, providing increased taxonomic resolution, more objective identifications, potential cost reductions, and reduced processing times. The use of DNA metabarcoding of bulk samples and environmental DNA (eDNA) is now widespread but is not yet universally implemented in national monitoring programs. While bulk sample metabarcoding involves extraction of DNA from organisms in a sample, eDNA analysis involves obtaining DNA directly from environmental samples, which can include microorganisms, meiofauna-size taxa and macrofauna traces such as larval stages, skin and hair cells, gametes, faeces and free DNA bound to particles. In Norway, freshwater biomonitoring in compliance with the EU Water Framework Directive (WFD) is conducted on several administrative levels, including national monitoring programs for running water, small and large lakes. These programs typically focus on a fraction of the actual biodiversity present in the monitored habitats (Weigand 2019). DNA metabarcoding of both bulk samples and eDNA samples are relevant tools for future freshwater biomonitoring in Norway. The aim of this PhD project is to develop assessment protocols based on DNA-metabarcoding and eDNA of benthic invertebrates, microcrustaceans and fish that can be used as standard biomonitoring tools to assess the ecological condition of lakes. The main topics addressed will be: - Development of protocols throughout the eDNA-metabarcoding workflow (i.e. sampling, filtration, preservation, extraction, amplification and sequencing) suitable to execute biodiversity assessments and determine the ecological status of lakes. - Comparison of the results obtained using molecular tools and traditional morphology-based approaches in order to assess the feasibility of such techniques to be incorporated as standard biomonitoring tools, such as the ones implemented under the provisions of the WFD. - Evaluate the effect of improved taxonomic resolution from molecular techniques on determining the ecological status of lakes, both by broadening the number of taxa analyzed and by identifying more taxa to species level. - Assess the feasibility of using eDNA extracted from water samples, taken at different depths and fish densities, to measure fish abundance/biomass as a proxy to calculate the ecological quality indices regulated in the WFD. - Analyze the coverage and resolution provided by reference libraries for certain taxa, such as crustacea, in order to assess the reliability and precision of taxonomic assignments.

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.

The Geographical Matrix

2009 ◽  
Author(s):  
A. D. Cliff ◽  
M.R. Smallman-Raynor ◽  
P. Haggett ◽  
D.F. Stroup ◽  
S.B. Thacker
Keyword(s):  
Public Health ◽  
Disease Surveillance ◽  
Communicable Disease ◽  
Communicable Diseases ◽  
League Of Nations ◽  
Morbidity And Mortality ◽  
Significant Public Health ◽  
Degree Of Certainty ◽  
The Times ◽  
Health Organization

A historical–geographical exploration of disease emergence is confronted by a series of fundamental questions: Which diseases have emerged? When? And where? For some high-profile diseases, such as Legionnaires’ disease, Ebola viral disease, and severe acute respiratory syndrome (SARS), the first recognized outbreaks are well documented in the scientific literature and the space–time coordinates of these early events can be fixed with a high degree of certainty. But, for some other diseases—especially those that, over the decades, have periodically resurfaced as significant public health problems—the times and places of their rise to prominence can be harder to specify. Accordingly, in this chapter we undertake a content analysis of three major epidemiological sources to identify patterns in the recognition and recording of communicable diseases of public health significance in the twentieth and early twenty-first centuries. Our analysis begins, in Section 3.2, with an examination of global and world regional patterns of communicable disease surveillance as documented in the annual statistical reports of the League of Nations/World Health Organization, 1923–83. In Section 3.3, we turn to the US Centers for Disease Control and Prevention’s (CDC’s) landmark publication Morbidity and Mortality Weekly Report (MMWR) to identify ‘headline trends’ in the national and international coverage of communicable diseases, 1952–2005. Finally, in Section 3.4, the inventory of epidemic assistance investigations (Epi-Aids) undertaken by CDC’s Epidemic Intelligence Service (EIS), 1946– 2005, provides a unique series of insights from the front line of epidemic investigative research. Informed by the evidence presented in these sections, Section 3.5 concludes by specifying the regional–thematic matrix of diseases for analysis in Chapters 4–9. The systematic international recording of information about morbidity and mortality from disease begins with the Health Organization of the League of Nations, established in the aftermath of the Great War. The first meeting of the Health Committee of the Health Section of the League took place in August 1921 to consider ‘the question of organising means of more rapid interchange of epidemiological information’ (Health Section of the League of Nations 1922: 3).

Sign in / Sign up

Export Citation Format

Share Document