Barcoding the food chain: from Sanger to high-throughput sequencing

Society faces the complex challenge of supporting biodiversity and ecosystem functioning, while ensuring food security by providing safe traceable food through an ever-more-complex global food chain. The increase in human mobility brings the added threat of pests, parasites, and invaders that further complicate our agro-industrial efforts. DNA barcoding technologies allow researchers to identify both individual species, and, when combined with universal primers and high-throughput sequencing techniques, the diversity within mixed samples (metabarcoding). These tools are already being employed to detect market substitutions, trace pests through the forensic evaluation of trace “environmental DNA”, and to track parasitic infections in livestock. The potential of DNA barcoding to contribute to increased security of the food chain is clear, but challenges remain in regulation and the need for validation of experimental analysis. Here, we present an overview of the current uses and challenges of applied DNA barcoding in agriculture, from agro-ecosystems within farmland to the kitchen table.

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Simultaneous absolute quantification and sequencing of fish environmental DNA in a mesocosm by quantitative sequencing technique

Scientific Reports ◽

10.1038/s41598-021-83318-6 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Tatsuhiko Hoshino ◽

Ryohei Nakao ◽

Hideyuki Doi ◽

Toshifumi Minamoto

Keyword(s):

Fish Species ◽

High Throughput Sequencing ◽

Correlation Coefficients ◽

Environmental Dna ◽

Digital Pcr ◽

Absolute Quantification ◽

Taqman Probe ◽

Individual Species ◽

Natural Environments ◽

Target Sequence

AbstractThe combination of high-throughput sequencing technology and environmental DNA (eDNA) analysis has the potential to be a powerful tool for comprehensive, non-invasive monitoring of species in the environment. To understand the correlation between the abundance of eDNA and that of species in natural environments, we have to obtain quantitative eDNA data, usually via individual assays for each species. The recently developed quantitative sequencing (qSeq) technique enables simultaneous phylogenetic identification and quantification of individual species by counting random tags added to the 5′ end of the target sequence during the first DNA synthesis. Here, we applied qSeq to eDNA analysis to test its effectiveness in biodiversity monitoring. eDNA was extracted from water samples taken over 4 days from aquaria containing five fish species (Hemigrammocypris neglectus, Candidia temminckii, Oryzias latipes, Rhinogobius flumineus, and Misgurnus anguillicaudatus), and quantified by qSeq and microfluidic digital PCR (dPCR) using a TaqMan probe. The eDNA abundance quantified by qSeq was consistent with that quantified by dPCR for each fish species at each sampling time. The correlation coefficients between qSeq and dPCR were 0.643, 0.859, and 0.786 for H. neglectus, O. latipes, and M. anguillicaudatus, respectively, indicating that qSeq accurately quantifies fish eDNA.

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High‐Throughput Sequencing of PCR Products Tagged with Universal Primers Using 454 Life Sciences Systems

Current Protocols in Molecular Biology ◽

10.1002/0471142727.mb0705s96 ◽

2011 ◽

Vol 96 (1) ◽

Cited By ~ 18

Author(s):

Derek Daigle ◽

Birgitte B. Simen ◽

Pascale Pochart

Keyword(s):

High Throughput ◽

High Throughput Sequencing ◽

Life Sciences ◽

Universal Primers ◽

Pcr Products

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High-throughput metabarcoding characterises fungal endophyte diversity in the phyllosphere of a barley crop

Phytobiomes Journal ◽

10.1094/pbiomes-09-20-0066-r ◽

2021 ◽

Author(s):

Carla Bridget Milazzo ◽

Katherine Grace Zulak ◽

Mariano Jordi Muria-Gonzalez ◽

Darcy Jones ◽

Matthew Power ◽

...

Keyword(s):

High Throughput ◽

High Throughput Sequencing ◽

Large Subunit ◽

Fungal Endophytes ◽

Environmental Dna ◽

Fungal Endophyte ◽

Bioinformatics Pipeline ◽

Pests And Diseases ◽

Key Factor ◽

Agroecosystem Management

Over the last decade, the microbiome has received increasing attention as a key factor in macroorganism fitness. Sustainable pest management requires an understanding of the complex microbial endophyte communities existing symbiotically within plants and the way synthetic pesticides interact with them. Fungal endophytes are known to benefit plant growth and fitness and may deter pests and diseases. Recent advances in high-throughput sequencing (HTS) have enabled integrative microbiome studies especially in agricultural contexts. Here we profile the fungal endophyte community in the phyllosphere of two barley (Hordeum vulgare) cultivars exposed to two systemic foliar fungicides using metabarcoding, a HTS tool that constructs community profiles from environmental DNA (eDNA). We studied the fungal nuclear ribosomal large subunit (LSU) D2 and ITS2 DNA markers through a bioinformatics pipeline introduced here. We found 88 and 128 unique amplicon sequence variants (ASVs) using the D2 and ITS2 metabarcoding assays, respectively. With principal coordinate analysis (PCoA) and PERMANOVA, ASV diversity did not change in response to barley cultivar or fungicide treatment, however the community structure of unsprayed plants did change between two collection times eight days apart. The workflow described here can be applied to other microbiome studies in agriculture and we hope it encourages further research into crop microbiomes to improve agroecosystem management.

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Novel Universal Primers for Metabarcoding eDNA Surveys of Marine Mammals and Other Marine Vertebrates

10.1101/759746 ◽

2019 ◽

Author(s):

Elena Valsecchi ◽

Jonas Bylemans ◽

Simon J. Goodman ◽

Roberto Lombardi ◽

Ian Carr ◽

...

Keyword(s):

Marine Mammals ◽

High Throughput Sequencing ◽

Environmental Dna ◽

16S Rrna Genes ◽

Rrna Genes ◽

Taxonomic Resolution ◽

Universal Primers ◽

Marine Biodiversity ◽

Primer Sets ◽

Marine Megafauna

ABSTRACTMetabarcoding studies using environmental DNA (eDNA) and high throughput sequencing (HTS) are rapidly becoming an important tool for assessing and monitoring marine biodiversity, detecting invasive species, and supporting basic ecological research. Several barcode loci targeting teleost fish and elasmobranchs have previously been developed, but to date primer sets focusing on other marine megafauna, such as marine mammals have received less attention. Similarly, there have been few attempts to identify potentially ‘universal’ barcode loci which may be informative across multiple marine vertebrate Orders. Here we describe the design and validation of four new sets of primers targeting hypervariable regions of the vertebrate mitochondrial 12S and 16S rRNA genes, which have conserved priming sites across virtually all cetaceans, pinnipeds, elasmobranchs, boney fish, sea turtles and birds, and amplify fragments with consistently high levels of taxonomically diagnostic sequence variation. ‘In silico’ validation using the OBITOOLS software showed our new barcode loci outperformed most existing vertebrate barcode loci for taxon detection and resolution. We also evaluated sequence diversity and taxonomic resolution of the new barcode loci in 680 complete marine mammal mitochondrial genomes demonstrating that they are effective at resolving amplicons for most taxa to the species level. Finally, we evaluated the performance of the primer sets with eDNA samples from aquarium communities with known species composition. These new primers will potentially allow surveys of complete marine vertebrate communities in single HTS metabarcoding assessments, simplifying workflows, reducing costs, and increasing accessibility to a wider range of investigators.

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GenBank is a reliable resource for 21st century biodiversity research

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1911714116 ◽

2019 ◽

Vol 116 (45) ◽

pp. 22651-22656 ◽

Cited By ~ 34

Author(s):

Matthieu Leray ◽

Nancy Knowlton ◽

Shian-Lei Ho ◽

Bryan N. Nguyen ◽

Ryuji J. Machida

Keyword(s):

Phylogenetic Analysis ◽

Global Change ◽

High Throughput ◽

High Throughput Sequencing ◽

Environmental Dna ◽

Taxonomic Resolution ◽

Biodiversity Research ◽

Animal Diversity ◽

Mitochondrial Sequences ◽

The Many

Traditional methods of characterizing biodiversity are increasingly being supplemented and replaced by approaches based on DNA sequencing alone. These approaches commonly involve extraction and high-throughput sequencing of bulk samples from biologically complex communities or samples of environmental DNA (eDNA). In such cases, vouchers for individual organisms are rarely obtained, often unidentifiable, or unavailable. Thus, identifying these sequences typically relies on comparisons with sequences from genetic databases, particularly GenBank. While concerns have been raised about biases and inaccuracies in laboratory and analytical methods, comparatively little attention has been paid to the taxonomic reliability of GenBank itself. Here we analyze the metazoan mitochondrial sequences of GenBank using a combination of distance-based clustering and phylogenetic analysis. Because of their comparatively rapid evolutionary rates and consequent high taxonomic resolution, mitochondrial sequences represent an invaluable resource for the detection of the many small and often undescribed organisms that represent the bulk of animal diversity. We show that metazoan identifications in GenBank are surprisingly accurate, even at low taxonomic levels (likely <1% error rate at the genus level). This stands in contrast to previously voiced concerns based on limited analyses of particular groups and the fact that individual researchers currently submit annotated sequences to GenBank without significant external taxonomic validation. Our encouraging results suggest that the rapid uptake of DNA-based approaches is supported by a bioinformatic infrastructure capable of assessing both the losses to biodiversity caused by global change and the effectiveness of conservation efforts aimed at slowing or reversing these losses.

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In silico assessment of primers for eDNA studies using PrimerTree and application to characterize the biodiversity surrounding the Cuyahoga River

10.1101/027235 ◽

2015 ◽

Cited By ~ 1

Author(s):

M.V. Cannon ◽

J. Hester ◽

A. Shalkhauser ◽

E.R. Chan ◽

K. Logue ◽

...

Keyword(s):

High Throughput ◽

Dna Sequences ◽

Water Samples ◽

High Throughput Sequencing ◽

Biological Diversity ◽

Pcr Amplification ◽

Environmental Dna ◽

Asian Carp ◽

Broad Perspective ◽

Wide Range

Analysis of environmental DNA (eDNA) enables the detection of species of interest from water and soil samples, typically using species-specific PCR. Here, we describe a method to characterize the biodiversity of a given environment by amplifying eDNA using primer pairs targeting a wide range of taxa and high-throughput sequencing for species identification. We tested this approach on 91 water samples of 40 mL collected along the Cuyahoga River (Ohio, USA). We amplified eDNA using 12 primer pairs targeting mammals, fish, amphibians, birds, bryophytes, arthropods, copepods, plants and several microorganism taxa and sequenced all PCR products simultaneously by high-throughput sequencing. Overall, we identified DNA sequences from 15 species of fish, 17 species of mammals, 8 species of birds, 15 species of arthropods, one turtle and one salamander. Interestingly, in addition to aquatic and semiaquatic animals, we identified DNA from terrestrial species that live near the Cuyahoga River. We also identified DNA from one Asian carp species invasive to the Great Lakes but that had not been previously reported in the Cuyahoga River. Our study shows that analysis of eDNA extracted from small water samples using wide-range PCR amplification combined with high-throughput sequencing can provide a broad perspective on biological diversity.

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Perspectives and benefits of high-throughput long-read sequencing in microbial ecology

Applied and Environmental Microbiology ◽

10.1128/aem.00626-21 ◽

2021 ◽

Author(s):

Leho Tedersoo ◽

Mads Albertsen ◽

Sten Anslan ◽

Benjamin Callahan

Keyword(s):

Microbial Ecology ◽

High Throughput ◽

Single Molecule ◽

High Throughput Sequencing ◽

Environmental Dna ◽

Nanopore Sequencing ◽

High Quality ◽

Short Read ◽

Sequencing Technologies ◽

Long Read

Short-read, high-throughput sequencing (HTS) methods have yielded numerous important insights into microbial ecology and function. Yet, in many instances short-read HTS techniques are suboptimal, for example by providing insufficient phylogenetic resolution or low integrity of assembled genomes. Single-molecule and synthetic long-read (SLR) HTS methods have successfully ameliorated these limitations. In addition, nanopore sequencing has generated a number of unique analysis opportunities such as rapid molecular diagnostics and direct RNA sequencing, and both PacBio and nanopore sequencing support detection of epigenetic modifications. Although initially suffering from relatively low sequence quality, recent advances have greatly improved the accuracy of long read sequencing technologies. In spite of great technological progress in recent years, the long-read HTS methods (PacBio and nanopore sequencing) are still relatively costly, require large amounts of high-quality starting material, and commonly need specific solutions in various analysis steps. Despite these challenges, long-read sequencing technologies offer high-quality, cutting-edge alternatives for testing hypotheses about microbiome structure and functioning as well as assembly of eukaryote genomes from complex environmental DNA samples.

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International Barcode of Life: Focus on big biodiversity in South Africa

Genome ◽

10.1139/gen-2017-0210 ◽

2017 ◽

Vol 60 (11) ◽

pp. 875-879 ◽

Cited By ~ 3

Author(s):

Sarah J. Adamowicz ◽

Peter M. Hollingsworth ◽

Sujeevan Ratnasingham ◽

Michelle van der Bank

Keyword(s):

South Africa ◽

Dna Barcoding ◽

High Throughput ◽

High Throughput Sequencing ◽

Sequence Data ◽

Global Scale ◽

Species Interaction ◽

Full Potential ◽

Data Generation ◽

African Flora

Participants in the 7th International Barcode of Life Conference (Kruger National Park, South Africa, 20–24 November 2017) share the latest findings in DNA barcoding research and its increasingly diversified applications. Here, we review prevailing trends synthesized from among 429 invited and contributed abstracts, which are collated in this open-access special issue of Genome. Hosted for the first time on the African continent, the 7th Conference places special emphasis on the evolutionary origins, biogeography, and conservation of African flora and fauna. Within Africa and elsewhere, DNA barcoding and related techniques are being increasingly used for wildlife forensics and for the validation of commercial products, such as medicinal plants and seafood species. A striking trend of the conference is the dramatic rise of studies on environmental DNA (eDNA) and on diverse uses of high-throughput sequencing techniques. Emerging techniques in these areas are opening new avenues for environmental biomonitoring, managing species-at-risk and invasive species, and revealing species interaction networks in unprecedented detail. Contributors call for the development of validated community standards for high-throughput sequence data generation and analysis, to enable the full potential of these methods to be realized for understanding and managing biodiversity on a global scale.

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Expedited assessment of terrestrial arthropod diversity by coupling Malaise traps with DNA barcoding

Genome ◽

10.1139/gen-2018-0093 ◽

2019 ◽

Vol 62 (3) ◽

pp. 85-95 ◽

Cited By ~ 12

Author(s):

Jeremy R. deWaard ◽

Valerie Levesque-Beaudin ◽

Stephanie L. deWaard ◽

Natalia V. Ivanova ◽

Jaclyn T.A. McKeown ◽

...

Keyword(s):

Dna Barcoding ◽

High Throughput ◽

National Park ◽

High Throughput Sequencing ◽

Species Abundance ◽

Arthropod Diversity ◽

Sequencing Technologies ◽

Data Release ◽

Speed And Accuracy ◽

Malaise Traps

Monitoring changes in terrestrial arthropod communities over space and time requires a dramatic increase in the speed and accuracy of processing samples that cannot be achieved with morphological approaches. The combination of DNA barcoding and Malaise traps allows expedited, comprehensive inventories of species abundance whose cost will rapidly decline as high-throughput sequencing technologies advance. Aside from detailing protocols from specimen sorting to data release, this paper describes their use in a survey of arthropod diversity in a national park that examined 21 194 specimens representing 2255 species. These protocols can support arthropod monitoring programs at regional, national, and continental scales.

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