Metabarcoding with MinION: Speeding up the detection of invasive aquatic species using environmental DNA and nanopore sequencing

AbstractTraditional detection of aquatic invasive species, via morphological identification is often time-consuming and can require a high level of taxonomic expertise, leading to delayed mitigation responses. Environmental DNA (eDNA) detection approaches of multiple species using Illumina-based sequencing technology have been used to overcome these hindrances, but sample processing is often lengthy. More recently, portable nanopore sequencing technology has become available, which has the potential to make molecular detection of invasive species more widely accessible and to substantially decrease sample turnaround times. However, nanopore-sequenced reads have a much higher error rate than those produced by Illumina platforms, which has so far hindered the adoption of this technology. We provide a detailed laboratory protocol and bioinformatic tools to increase the reliability of nanopore sequencing to detect invasive species, and we test its application using invasive bivalves. We sampled water from sites with pre-existing bivalve occurrence and abundance data, and contrasting bivalve communities, in Italy and Portugal. We extracted, amplified and sequenced eDNA with a turnaround of 3.5 days. The majority of processed reads were ≥ 99 % identical to reference sequences. There were no taxa detected other than those known to occur. The lack of detections of some species at some sites could be explained by their known low abundances. This is the first reported use of MinION to detect aquatic invasive species from eDNA samples. The approach can be easily adapted for other metabarcoding applications, such as biodiversity assessment, ecosystem health assessment and diet studies.

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Targeting the invaders – targeted detection of four priority freshwater invasive non-native species using environmental DNA

10.7287/peerj.preprints.27284 ◽

2018 ◽

Author(s):

Rosetta C Blackman ◽

Marco Benucci ◽

Robert Donnelly ◽

Bernd Hänfling ◽

Lynsey R Harper ◽

...

Keyword(s):

Invasive Species ◽

Native Species ◽

Environmental Dna ◽

Single Species ◽

Pcr Primers ◽

Field Trials ◽

Morphological Identification ◽

Mussel Species ◽

The Uk ◽

Monitoring Protocols

Early detection is paramount for attempts to remove invasive non-native species (INNS). Traditional methods rely on physical sampling and morphological identification, which can be problematic when species are in low densities and/or are cryptic. The use of environmental DNA (eDNA) as a monitoring tool in freshwater systems is becoming increasingly acceptable and widely used for the detection of single species. Here we demonstrate the development and application of standard PCR primers for the detection of four freshwater invasive species which are high priority for monitoring in the UK and elsewhere: Dreissenid mussels; Dreissena rostriformis bugensis (Andrusov, 1987) and D. polymorpha (Pallas, 1771), and Gammarid shrimps; Dikerogammarus villosus (Sowinsky, 1984) and D. haemobaphes (Eichwald, 1843). We carried out a rigorous validation process for testing the new primers, including DNA detection and degradation rate experiments in mesocosm, and a field comparison with traditional monitoring protocols. We successfully detected all four target species in mesocosms, but success was higher for mussels than shrimps. eDNA from single individuals of both mussel species could be detected within four hours of the start of the experiment. By contrast, shrimp were only consistently detected at higher densities (20 individuals). In field trials, the two mussel species and D. haemobaphes were detected at all sites where the species are known to be present, and eDNA consistently outperformed traditional kick sampling for species detection. However, D. villosus eDNA was only detected in one of five sites where the species was confirmed by kick sampling. These results demonstrate the applicability of standard PCR for eDNA detection of freshwater invasive species, but also highlight the importance of differences between taxa in terms of the detection ability.

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Targeting the invaders – targeted detection of four priority freshwater invasive non-native species using environmental DNA

10.7287/peerj.preprints.27284v1 ◽

2018 ◽

Author(s):

Rosetta C Blackman ◽

Marco Benucci ◽

Robert Donnelly ◽

Bernd Hänfling ◽

Lynsey R Harper ◽

...

Keyword(s):

Invasive Species ◽

Native Species ◽

Environmental Dna ◽

Single Species ◽

Pcr Primers ◽

Field Trials ◽

Morphological Identification ◽

Mussel Species ◽

The Uk ◽

Monitoring Protocols

Early detection is paramount for attempts to remove invasive non-native species (INNS). Traditional methods rely on physical sampling and morphological identification, which can be problematic when species are in low densities and/or are cryptic. The use of environmental DNA (eDNA) as a monitoring tool in freshwater systems is becoming increasingly acceptable and widely used for the detection of single species. Here we demonstrate the development and application of standard PCR primers for the detection of four freshwater invasive species which are high priority for monitoring in the UK and elsewhere: Dreissenid mussels; Dreissena rostriformis bugensis (Andrusov, 1987) and D. polymorpha (Pallas, 1771), and Gammarid shrimps; Dikerogammarus villosus (Sowinsky, 1984) and D. haemobaphes (Eichwald, 1843). We carried out a rigorous validation process for testing the new primers, including DNA detection and degradation rate experiments in mesocosm, and a field comparison with traditional monitoring protocols. We successfully detected all four target species in mesocosms, but success was higher for mussels than shrimps. eDNA from single individuals of both mussel species could be detected within four hours of the start of the experiment. By contrast, shrimp were only consistently detected at higher densities (20 individuals). In field trials, the two mussel species and D. haemobaphes were detected at all sites where the species are known to be present, and eDNA consistently outperformed traditional kick sampling for species detection. However, D. villosus eDNA was only detected in one of five sites where the species was confirmed by kick sampling. These results demonstrate the applicability of standard PCR for eDNA detection of freshwater invasive species, but also highlight the importance of differences between taxa in terms of the detection ability.

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Forage palatability of Broussonetia papyrifera an invasive species in Ghana: Relative preference and palatability by sheep and goats

Journal of Energy and Natural Resource Management ◽

10.26796/jenrm.v2i0.46 ◽

2018 ◽

Vol 2 (2) ◽

pp. 63-70 ◽

Cited By ~ 1

Author(s):

R. Obour

Keyword(s):

Invasive Species ◽

Leucaena Leucocephala ◽

Condensed Tannin ◽

Block Design ◽

Wet Season ◽

Broussonetia Papyrifera ◽

Sheep And Goats ◽

Relative Preference ◽

Exotic Tree ◽

High Level

Broussonetia papyrifera is an exotic tree widely grown for paper production. Due to its prolific regeneration it has invaded forestcanopy gaps and degraded farmlands and has now become an invasive species in Ghana. In enhancing its value for use the plantwas evaluated as potential forage for grazing animals vis-à-vis other two existing forage plants: Ficus exasperata and Leucaenaleucocephala.The study assessed the palatability and preference of Broussonetia papyrifera using sheep and goats for the wet anddry seasons.The species were assessed in indoor pen feeding trials using eight-unit (3×3 m) pens with the cafeteria method.The amount of forage offered was 100g (fresh material) in all instances for each species and for ten minutes. Adesign basedon 3×2×2 factorial in Randomized Complete Block Design (RCBD) was used to test the differences in palatability betweenthe three forage species.Results revealed that palatability was higher (P<0.05) in Leucaena leucocephala compared with Ficusexasperata and Broussonetia papyrifera for sheep and goats across seasons. The trend shown might be the result of the effectsof familiarity with the Leucaena leucocephala since animals tend to select plants that are familiar than newly introduced andunfamiliar plants. The study also revealed high level of condensed tannin (CT) in Broussonetia papyrifera which might haveinterfered with forage intake by the animals.There were no significant differences in palatability of Broussonetia papyrifera forgoat in both dry and wet season interactions and Ficus exasperata for goat in both dry and wet season interactions (P>0.05).Thestudy concluded that Broussonetia papyrifera could be a potential feed for both sheep and goats across seasons.The researchrecommended that livestock farmers should incorporate Broussonetia papyrifera feed into their programmes for both sheep andgoats and should be introduced to animals from infancy so that it may become a familiar feed for them.

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Long Term Interactions of Native and Invasive Species in a Marine Protected Area Suggest Complex Cascading Effects Challenging Conservation Outcomes

Diversity ◽

10.3390/d13020071 ◽

2021 ◽

Vol 13 (2) ◽

pp. 71

Author(s):

Charalampos Dimitriadis ◽

Ivoni Fournari-Konstantinidou ◽

Laurent Sourbès ◽

Drosos Koutsoubas ◽

Stelios Katsanevakis

Keyword(s):

Invasive Species ◽

Native Species ◽

High Performance ◽

Marine Protected Area ◽

Habitat Degradation ◽

Population Increase ◽

Rocky Reef ◽

Cascading Effects ◽

High Level

Understanding the interactions among invasive species, native species and marine protected areas (MPAs), and the long-term regime shifts in MPAs is receiving increased attention, since biological invasions can alter the structure and functioning of the protected ecosystems and challenge conservation efforts. Here we found evidence of marked modifications in the rocky reef associated biota in a Mediterranean MPA from 2009 to 2019 through visual census surveys, due to the presence of invasive species altering the structure of the ecosystem and triggering complex cascading effects on the long term. Low levels of the populations of native high-level predators were accompanied by the population increase and high performance of both native and invasive fish herbivores. Subsequently the overgrazing and habitat degradation resulted in cascading effects towards the diminishing of the native and invasive invertebrate grazers and omnivorous benthic species. Our study represents a good showcase of how invasive species can coexist or exclude native biota and at the same time regulate or out-compete other established invaders and native species.

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Nanopore sequencing technology and tools for genome assembly: computational analysis of the current state, bottlenecks and future directions

Briefings in Bioinformatics ◽

10.1093/bib/bby017 ◽

2018 ◽

Vol 20 (4) ◽

pp. 1542-1559 ◽

Cited By ~ 44

Author(s):

Damla Senol Cali ◽

Jeremie S Kim ◽

Saugata Ghose ◽

Can Alkan ◽

Onur Mutlu

Keyword(s):

Sequence Analysis ◽

Genome Assembly ◽

Sequence Data ◽

Error Rates ◽

Nanopore Sequencing ◽

Memory Usage ◽

Sequencing Technology ◽

Assembly Pipeline ◽

And Performance ◽

Polishing Tool

Abstract Nanopore sequencing technology has the potential to render other sequencing technologies obsolete with its ability to generate long reads and provide portability. However, high error rates of the technology pose a challenge while generating accurate genome assemblies. The tools used for nanopore sequence analysis are of critical importance, as they should overcome the high error rates of the technology. Our goal in this work is to comprehensively analyze current publicly available tools for nanopore sequence analysis to understand their advantages, disadvantages and performance bottlenecks. It is important to understand where the current tools do not perform well to develop better tools. To this end, we (1) analyze the multiple steps and the associated tools in the genome assembly pipeline using nanopore sequence data, and (2) provide guidelines for determining the appropriate tools for each step. Based on our analyses, we make four key observations: (1) the choice of the tool for basecalling plays a critical role in overcoming the high error rates of nanopore sequencing technology. (2) Read-to-read overlap finding tools, GraphMap and Minimap, perform similarly in terms of accuracy. However, Minimap has a lower memory usage, and it is faster than GraphMap. (3) There is a trade-off between accuracy and performance when deciding on the appropriate tool for the assembly step. The fast but less accurate assembler Miniasm can be used for quick initial assembly, and further polishing can be applied on top of it to increase the accuracy, which leads to faster overall assembly. (4) The state-of-the-art polishing tool, Racon, generates high-quality consensus sequences while providing a significant speedup over another polishing tool, Nanopolish. We analyze various combinations of different tools and expose the trade-offs between accuracy, performance, memory usage and scalability. We conclude that our observations can guide researchers and practitioners in making conscious and effective choices for each step of the genome assembly pipeline using nanopore sequence data. Also, with the help of bottlenecks we have found, developers can improve the current tools or build new ones that are both accurate and fast, to overcome the high error rates of the nanopore sequencing technology.

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Development of species-specific Cichla species eDNA primers for alien invasive species (AIS) monitoring in Malaysia

ARPHA Conference Abstracts ◽

10.3897/aca.4.e65449 ◽

2021 ◽

Vol 4 ◽

Author(s):

O. Nurul Fizatul Nabilah ◽

A. R. Ramizah ◽

A. B. Adibah ◽

S. Syazwan ◽

A.G. Intan Faraha ◽

...

Keyword(s):

Dna Sequences ◽

Environmental Dna ◽

Coi Gene ◽

Alien Invasive Species ◽

Survey Method ◽

Specific Primers ◽

Invasive Fishes ◽

Specific Amplification ◽

Species Specific ◽

High Level

Peacock bass or the cichlids are known locally as top predator fishes which are invasive in Malaysia freshwater system. Detection probabilities for these fishes are typically low, especially using conventional capture-survey method due to the fish’s behaviour of hiding beneath the water’s surface. Hence, the environmental DNA (eDNA) monitoring is a relatively new approach that can be used to assess the distribution of these invasive fishes. Here, we report the strategy to develop small fragment (280- 400 bp) specific-specific primers for three selected invasive Cichla species namely, C. ocellaris, C. monoculus, and C. kelberi based on mitochondrial DNA (mtDNA) sequences. Current research showed that the developed species-specific primers from cytochrome oxidase I (COI) gene has high resolution at species level. Species-specific amplification tests also proved the specificity of the developed primers, securing the high- level species identification potential which may help in controlling the spread of alien invasive fish species.

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Use of environmental DNA (eDNA) in streams to detect feral swine (Sus scrofa)

PeerJ ◽

10.7717/peerj.8287 ◽

2020 ◽

Vol 8 ◽

pp. e8287

Author(s):

Amberly N. Hauger ◽

Karmen M. Hollis-Etter ◽

Dwayne R. Etter ◽

Gary J. Roloff ◽

Andrew R. Mahon

Keyword(s):

Invasive Species ◽

Water Temperature ◽

Linear Model ◽

Detection Probability ◽

Generalized Linear Model ◽

Environmental Dna ◽

The United States ◽

Stream Data ◽

Feral Swine ◽

Animal Populations

Invasive feral swine can damage ecosystems, disrupt plant and animal populations, and transmit diseases. Monitoring of feral swine populations requires expensive and labor-intensive techniques such as aerial surveys, field surveys for sign, trail cameras, and verifying landowner reports. Environmental DNA (eDNA) provides an alternative method for locating feral swine. To aid in detection of this harmful invasive species, a novel assay was developed incorporating molecular methods. From August 2017 to April 2018, water samples and stream data were collected along 400 m transects in two different stream types where swine DNA was artificially introduced to investigate potential factors affecting detection. A generalized linear model (family binomial) was used to characterize environmental conditions affecting swine DNA detection; detection was the dependent variable and stream measurements included stream type, distance downstream, water temperature, velocity, turbidity, discharge, and pH as independent variables. Parameters from the generalized linear model were deemed significant if 95% confidence intervals did not overlap 0. Detection probability for swine DNA negatively related to water temperature (β = − 0.21, 95% CI [−0.35 to −0.09]), with the highest detection probability (0.80) at 0 °C and lowest detection probability (0.05) at 17.9 °C water temperature. Results indicate that sampling for swine eDNA in free-flowing stream systems should occur at lower water temperatures to maximize detection probability. This study provides a foundation for further development of field and sampling techniques for utilizing eDNA as a viable alternative to monitoring a terrestrial invasive species in northern regions of the United States.

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Microbial Eukaryotes: a Missing Link in Gut Microbiome Studies

mSystems ◽

10.1128/msystems.00201-17 ◽

2018 ◽

Vol 3 (2) ◽

Cited By ~ 28

Author(s):

Isabelle Laforest-Lapointe ◽

Marie-Claire Arrieta

Keyword(s):

Microbial Communities ◽

Microbial Ecology ◽

High Throughput Sequencing ◽

Sequencing Technology ◽

Human Gut ◽

Microbial Eukaryotes ◽

Host Health ◽

High Level ◽

Eukaryotic Organisms ◽

New Evidence

ABSTRACTHuman-associated microbial communities include prokaryotic and eukaryotic organisms across high-level clades of the tree of life. While advances in high-throughput sequencing technology allow for the study of diverse lineages, the vast majority of studies are limited to bacteria, and very little is known on how eukaryote microbes fit in the overall microbial ecology of the human gut. As recent studies consider eukaryotes in their surveys, it is becoming increasingly clear that eukaryotes play important ecological roles in the microbiome as well as in host health. In this perspective, we discuss new evidence on eukaryotes as fundamental species of the human gut and emphasize that future microbiome studies should characterize the multitrophic interactions between microeukaryotes, other microorganisms, and the host.

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