scholarly journals Evaluating metabarcoding to analyse diet composition of species foraging in anthropogenic landscapes using Ion Torrent and Illumina sequencing

2018 ◽  
Author(s):  
Marie-Amélie Forin-Wiart ◽  
Marie-Lazarine Poulle ◽  
Sylvain Piry ◽  
Jean-François Cosson ◽  
Claire Larose ◽  
...  
Keyword(s):  
High Throughput Sequencing ◽  
Pet Food ◽  
Anthropogenic Landscapes ◽  
Invasive Approach ◽  
Food Items ◽  
Wide Range ◽  
Dna Metabarcoding ◽  
Pcr Duplicates ◽  
Ion Pgm ◽  
Sequence Quality

AbstractDNA metabarcoding of faecal samples is being successfully used to study the foraging niche of species. We assessed the ability of two benchtop high-throughput sequencing (HTS) platforms, to identify a large taxonomic array of food items from domestic cats Felis silvestris catus, including prey and human-related food taxa (pet food and leftovers leaving undetectable solid remains in faeces). Scats from a captive feeding trial (n=41) and from free-ranging individuals (n=326) were collected and analysed using a cytb mini-barcode in independent PCR duplicates on the Ion PGM and the MiSeq platforms. Outputs from MiSeq were more sensitive and reproducible than those from Ion PGM due to a higher sequencing depth and sequence quality on MiSeq. DNA from intact prey taxa was detected more often (82% of the expected occurrences) than DNA from pet food (54%) and raw fish and meat (31%). We assumed that this variability was linked to different degree of DNA degradation: The Ion PGM detected significantly less human-linked food, birds, field voles, murids and shrews in the field-collected samples than the MiSeq platform. Pooling the replicates from both platforms and filtering the data allowed identification of at least one food item in 87.4% of the field-collected samples. Our DNA metabarcoding approach identified 29 prey taxa, of which 25 to species level (90% of items) including 9 rodents, 3 insectivores, 12 birds and 1 reptile and 33 human-related food taxa of which 23 were identified to genus level (75% of items). Our results demonstrate that using HTS platforms such as MiSeq, which provide reads of sufficiently high quantity and quality, with sufficient numbers of technical replicates, is a robust and non-invasive approach for further dietary studies on animals foraging on a wide range of food items in anthropogenic landscapes.

scholarly journals Validation and standardization of DNA extraction and library construction methods for metagenomics-based human fecal microbiome measurements

Microbiome ◽  
2021 ◽  
Vol 9 (1) ◽  
Author(s):  
Dieter M. Tourlousse ◽  
Koji Narita ◽  
Takamasa Miura ◽  
Mitsuo Sakamoto ◽  
Akiko Ohashi ◽  
...  
Keyword(s):  
Dna Extraction ◽  
High Throughput Sequencing ◽  
Performance Metrics ◽  
Collaborative Study ◽  
Human Microbiome ◽  
Second Phase ◽  
Intermediate Precision ◽  
Fecal Microbiome ◽  
Library Construction ◽  
Wide Range

Abstract Background Validation and standardization of methodologies for microbial community measurements by high-throughput sequencing are needed to support human microbiome research and its industrialization. This study set out to establish standards-based solutions to improve the accuracy and reproducibility of metagenomics-based microbiome profiling of human fecal samples. Results In the first phase, we performed a head-to-head comparison of a wide range of protocols for DNA extraction and sequencing library construction using defined mock communities, to identify performant protocols and pinpoint sources of inaccuracy in quantification. In the second phase, we validated performant protocols with respect to their variability of measurement results within a single laboratory (that is, intermediate precision) as well as interlaboratory transferability and reproducibility through an industry-based collaborative study. We further ascertained the performance of our recommended protocols in the context of a community-wide interlaboratory study (that is, the MOSAIC Standards Challenge). Finally, we defined performance metrics to provide best practice guidance for improving measurement consistency across methods and laboratories. Conclusions The validated protocols and methodological guidance for DNA extraction and library construction provided in this study expand current best practices for metagenomic analyses of human fecal microbiota. Uptake of our protocols and guidelines will improve the accuracy and comparability of metagenomics-based studies of the human microbiome, thereby facilitating development and commercialization of human microbiome-based products.

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.

ViDiT–CACTUS: an inexpensive and versatile library preparation and sequence analysis method for virus discovery and other microbiology applications

2018 ◽  
Vol 64 (10) ◽  
pp. 761-773 ◽  
Author(s):  
Joost T.P. Verhoeven ◽  
Marta Canuti ◽  
Hannah J. Munro ◽  
Suzanne C. Dufour ◽  
Andrew S. Lang
Keyword(s):  
Influenza A ◽  
High Throughput Sequencing ◽  
Low Cost ◽  
Quality Data ◽  
Library Preparation ◽  
Virus Discovery ◽  
Influenza A Viruses ◽  
Wide Range ◽  
Functional Profiling ◽  
Biology Laboratory

High-throughput sequencing (HTS) technologies are becoming increasingly important within microbiology research, but aspects of library preparation, such as high cost per sample or strict input requirements, make HTS difficult to implement in some niche applications and for research groups on a budget. To answer these necessities, we developed ViDiT, a customizable, PCR-based, extremely low-cost (less than US$5 per sample), and versatile library preparation method, and CACTUS, an analysis pipeline designed to rely on cloud computing power to generate high-quality data from ViDiT-based experiments without the need of expensive servers. We demonstrate here the versatility and utility of these methods within three fields of microbiology: virus discovery, amplicon-based viral genome sequencing, and microbiome profiling. ViDiT–CACTUS allowed the identification of viral fragments from 25 different viral families from 36 oropharyngeal–cloacal swabs collected from wild birds, the sequencing of three almost complete genomes of avian influenza A viruses (>90% coverage), and the characterization and functional profiling of the complete microbial diversity (bacteria, archaea, viruses) within a deep-sea carnivorous sponge. ViDiT–CACTUS demonstrated its validity in a wide range of microbiology applications, and its simplicity and modularity make it easily implementable in any molecular biology laboratory, towards various research goals.

scholarly journals DNA metabarcoding of fungal diversity in air and snow of Livingston Island, South Shetland Islands, Antarctica

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Luiz Henrique Rosa ◽  
Otávio Henrique Bezerra Pinto ◽  
Tina Šantl-Temkiv ◽  
Peter Convey ◽  
Micheline Carvalho-Silva ◽  
...  
Keyword(s):  
Fungal Diversity ◽  
High Throughput Sequencing ◽  
Rank Order ◽  
Mesh Size ◽  
Diversity Indices ◽  
South Shetland Islands ◽  
Livingston Island ◽  
Dna Metabarcoding ◽  
Air Sample ◽  
Total Dna

AbstractWe assessed fungal diversity present in air and freshly deposited snow samples obtained from Livingston Island, Antarctica, using DNA metabarcoding through high throughput sequencing (HTS). A total of 740 m3 of air were pumped through a 0.22 µm membrane. Snow obtained shortly after deposition was kept at room temperature and yielded 3.760 L of water, which was filtered using Sterivex membranes of 0.22 µm mesh size. The total DNA present was extracted and sequenced. We detected 171 fungal amplicon sequence variants (ASVs), 70 from the air and 142 from the snow. They were dominated by the phyla Ascomycota, Basidiomycota, Mortierellomycota and Mucoromycota. Pseudogymnoascus, Cladosporium, Mortierella and Penicillium sp. were the most dominant ASVs detected in the air in rank order. In snow, Cladosporium, Pseudogymnoascus, Penicillium, Meyerozyma, Lecidea, Malassezia, Hanseniaspora, Austroplaca, Mortierella, Rhodotorula, Penicillium, Thelebolus, Aspergillus, Poaceicola, Glarea and Lecanora were the dominant ASVs present. In general, the two fungal assemblages displayed high diversity, richness, and dominance indices, with the assemblage found in snow having the highest diversity indices. Of the total fungal ASVs detected, 29 were only present in the air sample and 101 in the snow sample, with only 41 present in both samples; however, when only the dominant taxa from both samples were compared none occurred only in the air and, among the rare portion, 26 taxa occurred in both air and snow. Application of HTS revealed the presence of a more diverse fungal community in the air and snow of Livingston Island in comparison with studies using traditional isolation methods. The assemblages were dominated by cold-adapted and cosmopolitan fungal taxa, including members of the genera Pseudogymnoascus, Malassezia and Rhodotorula, which include some taxa reported as opportunistic. Our results support the hypothesis that the presence of microbiota in the airspora indicates the possibility of dispersal around Antarctica in the air column. However, further aeromycology studies are required to understand the dynamics of fungal dispersal within and beyond Antarctica.

scholarly journals Characterization of Stool Virome in Children Newly Diagnosed With Moderate to Severe Ulcerative Colitis

2019 ◽  
Vol 25 (10) ◽  
pp. 1656-1662 ◽  
Author(s):  
Rafal Tokarz ◽  
Jeffrey S Hyams ◽  
David R Mack ◽  
Brendan Boyle ◽  
Anne M Griffiths ◽  
...  
Keyword(s):  
Ulcerative Colitis ◽  
High Throughput Sequencing ◽  
Viral Infections ◽  
Control Group ◽  
Newly Diagnosed ◽  
Control Cohort ◽  
Fecal Samples ◽  
Wide Range ◽  
Irritable Bowel

Abstract Background Viral infections have been suggested as possible triggers for the onset of ulcerative colitis (UC). Methods We employed VirCapSeq-Vert, a high-throughput sequencing virus capture platform, to examine the stool virome of children with newly diagnosed moderate to severe UC. We surveyed fecal samples collected at presentation, after symptom remission, and from a control group diagnosed with irritable bowel syndrome. Results Seventy subjects with UC (mean age 13 years, 45 had moderate symptoms, 25 had severe, 69 of 70 had a Mayo endoscopy subscore 2/3) were studied. We detected a wide range of animal viruses that were taxonomically classified into 12 viral families. A virus was present in 50% of fecal samples collected at presentation, 41% of samples collected after remission, and 40% of samples in our control group. The most frequently identified viruses were diet-based gyroviruses. The UC cohort had a significantly higher prevalence of anelloviruses compared with the control cohort. However, we did not identify a single virus that can be implicated in the onset of UC and did not find an association between UC disease severity and viral presence. Conclusion Presence of virus in stool was not associated with the onset of pediatric UC.

scholarly journals Wild specimens of sand fly phlebotomine Lutzomyia evansi, vector of leishmaniasis, show high abundance of Methylobacterium and natural carriage of Wolbachia and Cardinium types in the midgut microbiome

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Rafael J. Vivero ◽  
Marcela Villegas-Plazas ◽  
Gloria E. Cadavid-Restrepo ◽  
Claudia Ximena Moreno Herrera ◽  
Sandra I. Uribe ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
High Throughput Sequencing ◽  
Sand Fly ◽  
Human Populations ◽  
Rrna Gene ◽  
Remarkable Feature ◽  
Core Microbiome ◽  
Bacterial Phyla ◽  
Wide Range

AbstractPhlebotomine sand flies are remarkable vectors of several etiologic agents (virus, bacterial, trypanosomatid Leishmania), posing a heavy health burden for human populations mainly located at developing countries. Their intestinal microbiota is involved in a wide range of biological and physiological processes, and could exclude or facilitate such transmission of pathogens. In this study, we investigated the Eubacterial microbiome from digestive tracts of Lu. evansi adults structure using 16S rRNA gene sequence amplicon high throughput sequencing (Illumina MiSeq) obtained from digestive tracts of Lu. evansi adults. The samples were collected at two locations with high incidence of the disease in humans: peri-urban and forest ecosystems from the department of Sucre, Colombia. 289,068 quality-filtered reads of V4 region of 16S rRNA gene were obtained and clustered into 1,762 operational taxonomic units (OTUs) with 97% similarity. Regarding eubacterial diversity, 14 bacterial phyla and 2 new candidate phyla were found to be consistently associated with the gut microbiome content. Proteobacteria, Firmicutes, and Bacteroidetes were the most abundant phyla in all the samples and the core microbiome was particularly dominated by Methylobacterium genus. Methylobacterium species, are known to have mutualistic relationships with some plants and are involved in shaping the microbial community in the phyllosphere. As a remarkable feature, OTUs classified as Wolbachia spp. were found abundant on peri-urban ecosystem samples, in adult male (OTUs n = 776) and unfed female (OTUs n = 324). Furthermore, our results provide evidence of OTUs classified as Cardinium endosymbiont in relative abundance, notably higher with respect to Wolbachia. The variation in insect gut microbiota may be determined by the environment as also for the type of feeding. Our findings increase the richness of the microbiota associated with Lu. evansi. In this study, OTUs of Methylobacterium found in Lu. evansi was higher in engorged females, suggesting that there are interactions between microbes from plant sources, blood nutrients and the parasites they transmit during the blood intake.

scholarly journals Accurate detection for a wide range of mutation and editing sites of microRNAs from small RNA high-throughput sequencing profiles

2016 ◽  
Vol 44 (14) ◽  
pp. e123-e123 ◽  
Author(s):  
Yun Zheng ◽  
Bo Ji ◽  
Renhua Song ◽  
Shengpeng Wang ◽  
Ting Li ◽  
...  
Keyword(s):  
High Throughput ◽  
Small Rna ◽  
High Throughput Sequencing ◽  
Accurate Detection ◽  
Wide Range

scholarly journals Vision and touch in relation to foraging and predator detection: insightful contrasts between a plover and a sandpiper

2008 ◽  
Vol 276 (1656) ◽  
pp. 437-445 ◽  
Author(s):  
Graham R Martin ◽  
Theunis Piersma
Keyword(s):  
Visual Fields ◽  
Skeletal Structure ◽  
Visually Guided ◽  
Sources Of Information ◽  
Predator Detection ◽  
Food Items ◽  
Binocular Field ◽  
Blind Area ◽  
Wide Range ◽  
Breeding Grounds

Visual fields were determined in two species of shorebirds (Charadriiformes) whose foraging is guided primarily by different sources of information: red knots ( Calidris canutus , tactile foragers) and European golden plovers ( Pluvialis apricaria , visual foragers). The visual fields of both species showed features that are found in a wide range of birds whose foraging involves precision pecking or lunging at food items. Surprisingly, red knots did not show comprehensive panoramic vision as found in some other tactile feeders; they have a binocular field surrounding the bill and a substantial blind area behind the head. We argue that this is because knots switch to more visually guided foraging on their breeding grounds. However, this visual field topography leaves them vulnerable to predation, especially when using tactile foraging in non-breeding locations where predation by falcons is an important selection factor. Golden plovers use visually guided foraging throughout the year, and so it is not surprising that they have precision-pecking frontal visual fields. However, they often feed at night and this is associated with relatively large eyes. These are anchored in the skull by a wing of bone extending from the dorsal perimeter of each orbit; a skeletal structure previously unreported in birds and which we have named ‘supraorbital aliform bone’, Os supraorbitale aliforme . The larger eyes and their associated supraorbital wings result in a wide blind area above the head, which may leave these plovers particularly vulnerable to predation. Thus, in these two shorebirds, we see clear examples of the trade-off between the two key functions of visual fields: (i) the detection of predators remote from the animal and (ii) the control of accurate behaviours, such as the procurement of food items, at close quarters.

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