Single Fragment Or Bulk Soil DNA Metabarcoding: Which Is Better for Characterizing Biological Taxa Found in Surface Soils for Sample Separation?
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DNA Metabarcoding of Microbial Communities for Healthcare I
Reviews ISSN 1993-6842 (on-line); ISSN 0233-7657 (print) Biopolymers and Cell. 2016. Vol. 32. N 1. P 3–8 doi: http://dx.doi.org/10.7124/bc.000906 UDC 577.25 + 579.61 DNA metabarcoding of microbial communities for healthcare I. Ye. Zaets1, O. V. Podolich1, O. N. Reva2, N. O. Kozyrovska1 1 Institute of Molecular Biology and Genetics, NAS of Ukraine, 150, Akademika Zabolotnoho Str., Kyiv, Ukraine, 03680 2 Department of Biochemistry, Bioinformatics and Computational Biology Unit, University of Pretoria Lynnwood road, Hillcrest, Pretoria, South Africa, 0002 [email protected], [email protected], [email protected] High-throughput sequencing allows obtaining DNA barcodes of multiple species of microorganisms from a single environmental sample. Next Generation Sequencing (NGS)-based profiling provides new opportunities to evaluate the human health effect of microbial community members affiliated to probiotics. DNA metabar- coding may serve as a quality control of microbial communities, comprising complex probiotics and other fermented foods. A detailed inventory of complex communities is a pre-requisite of understanding their func- tionality as whole entities that makes it possible to design more effective bio-products by precise replacement of one community member by others. The present paper illustrates how the NGS-based DNA metabarcoding allows profiling of both wild and hybrid multi-microbial communities with the example of a kombucha probi- otic beverage fermented by yeast-bacterial partners. Keywords: DNA metabarcoding, microbial communities, healthcare, -
Metagenomics Approaches for the Detection and Surveillance of Emerging and Recurrent Plant Pathogens
microorganisms Review Metagenomics Approaches for the Detection and Surveillance of Emerging and Recurrent Plant Pathogens Edoardo Piombo 1,2 , Ahmed Abdelfattah 3,4 , Samir Droby 5, Michael Wisniewski 6,7, Davide Spadaro 1,8,* and Leonardo Schena 9 1 Department of Agricultural, Forest and Food Sciences (DISAFA), University of Torino, 10095 Grugliasco, Italy; [email protected] 2 Department of Forest Mycology and Plant Pathology, Uppsala Biocenter, Swedish University of Agricultural Sciences, P.O. Box 7026, 75007 Uppsala, Sweden 3 Institute of Environmental Biotechnology, Graz University of Technology, Petersgasse 12, 8010 Graz, Austria; [email protected] 4 Department of Ecology, Environment and Plant Sciences, University of Stockholm, Svante Arrhenius väg 20A, 11418 Stockholm, Sweden 5 Department of Postharvest Science, Agricultural Research Organization (ARO), The Volcani Center, Rishon LeZion 7505101, Israel; [email protected] 6 U.S. Department of Agriculture—Agricultural Research Service (USDA-ARS), Kearneysville, WV 25430, USA; [email protected] 7 Department of Biological Sciences, Virginia Technical University, Blacksburg, VA 24061, USA 8 AGROINNOVA—Centre of Competence for the Innovation in the Agroenvironmental Sector, University of Torino, 10095 Grugliasco, Italy 9 Department of Agriculture, Università Mediterranea, 89122 Reggio Calabria, Italy; [email protected] * Correspondence: [email protected]; Tel.: +39-0116708942 Abstract: Globalization has a dramatic effect on the trade and movement of seeds, fruits and vegeta- bles, with a corresponding increase in economic losses caused by the introduction of transboundary Citation: Piombo, E.; Abdelfattah, A.; plant pathogens. Current diagnostic techniques provide a useful and precise tool to enact surveillance Droby, S.; Wisniewski, M.; Spadaro, protocols regarding specific organisms, but this approach is strictly targeted, while metabarcoding D.; Schena, L. -
Cristescu TREE 2014.Pdf
TREE-1853; No. of Pages 6 Opinion From barcoding single individuals to metabarcoding biological communities: towards an integrative approach to the study of global biodiversity Melania E. Cristescu Department of Biology, McGill University, Montreal, QC H3A 1B1, Canada DNA-based species identification, known as barcoding, introduced by Arnot et al. [6] and was firmly advanced transformed the traditional approach to the study of and standardized by Hebert et al. [7]. The simple idea of biodiversity science. The field is transitioning from bar- using a short DNA fragment as a barcode (see Glossary) for coding individuals to metabarcoding communities. This identifying species across the Metazoa has been both revolution involves new sequencing technologies, bio- strongly embraced and vigorously scrutinized over the past informatics pipelines, computational infrastructure, and decade [8,9]. Nevertheless, the efforts led by Paul Hebert, experimental designs. In this dynamic genomics land- and supported by the Consortium for the Barcode of Life scape, metabarcoding studies remain insular and biodi- (CBoL; http://www.barcodeoflife.org/) resulted in a global versity estimates depend on the particular methods enterprise that combined molecular tools with valuable but used. In this opinion article, I discuss the need for a scarce taxonomic expertise [10,11]. Today, DNA barcodes coordinated advancement of DNA-based species identi- are being used commonly to identify specimens and the fication that integrates taxonomic and barcoding infor- approach has wide applications in biodiversity conserva- mation. Such an approach would facilitate access to tion, environmental management, invasion biology, the almost 3 centuries of taxonomic knowledge and 1 de- study of trophic interactions, and food safety [12–14]. -
Edna in a Bottleneck: Obstacles to Fish Metabarcoding Studies in Megadiverse Freshwater 3 Systems 4 5 Authors: 6 Jake M
bioRxiv preprint doi: https://doi.org/10.1101/2021.01.05.425493; this version posted January 7, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. 1 Title: 2 eDNA in a bottleneck: obstacles to fish metabarcoding studies in megadiverse freshwater 3 systems 4 5 Authors: 6 Jake M. Jackman1, Chiara Benvenuto1, Ilaria Coscia1, Cintia Oliveira Carvalho2, Jonathan S. 7 Ready2, Jean P. Boubli1, William E. Magnusson3, Allan D. McDevitt1* and Naiara Guimarães 8 Sales1,4* 9 10 Addresses: 11 1Environment and Ecosystem Research Centre, School of Science, Engineering and Environment, 12 University of Salford, Salford, M5 4WT, UK 13 2Centro de Estudos Avançados de Biodiversidade, Instituto de Ciências Biológicas, Universidade 14 Federal do Pará, Belém, Brazil 15 3Coordenação de Biodiversidade, Instituto Nacional de Pesquisas da Amazônia, Manaus, 16 Amazonas, Brazil 17 4CESAM - Centre for Environmental and Marine Studies, Departamento de Biologia Animal, 18 Faculdade de Ciências da Universidade de Lisboa, Lisbon, Portugal 19 20 *Corresponding authors: 21 Naiara Guimarães Sales, [email protected] 22 Allan McDevitt, [email protected] 23 24 Running title: Obstacles to eDNA surveys in megadiverse systems 25 26 Keywords: Amazon, barcoding gap, freshwater, MiFish, Neotropics, reference database, 27 taxonomic resolution 28 1 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.05.425493; this version posted January 7, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. -
Metabarcoding in the Abyss: Uncovering Deep-Sea Biodiversity Through Environmental
Metabarcoding in the abyss : uncovering deep-sea biodiversity through environmental DNA Miriam Isabelle Brandt To cite this version: Miriam Isabelle Brandt. Metabarcoding in the abyss : uncovering deep-sea biodiversity through environmental DNA. Agricultural sciences. Université Montpellier, 2020. English. NNT : 2020MONTG033. tel-03197842 HAL Id: tel-03197842 https://tel.archives-ouvertes.fr/tel-03197842 Submitted on 14 Apr 2021 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. THÈSE POUR OBTENIR LE GRADE DE DOCTEUR DE L’UNIVERSITÉ DE M ONTPELLIER En Sciences de l'Évolution et de la Biodiversité École doctorale GAIA Unité mixte de recherche MARBEC Pourquoi Pas les Abysses ? L’ADN environnemental pour l’étude de la biodiversité des grands fonds marins Metabarcoding in the abyss: uncovering deep - sea biodiversity through environmental DNA Présentée par Miriam Isabelle BRANDT Le 10 juillet 2020 Sous la direction de Sophie ARNAUD-HAOND et Daniela ZEPPILLI Devant le jury composé de Sofie DERYCKE, Senior researcher/Professeur rang A, ILVO, Belgique Rapporteur -
Bacteriophage T7 DNA Polymerase – Sequenase
Bacteriophage T7 DNA polymerase – sequenase The Harvard community has made this article openly available. Please share how this access benefits you. Your story matters Citation Zhu, Bin. 2014. “Bacteriophage T7 DNA polymerase – sequenase.” Frontiers in Microbiology 5 (1): 181. doi:10.3389/fmicb.2014.00181. http://dx.doi.org/10.3389/fmicb.2014.00181. Published Version doi:10.3389/fmicb.2014.00181 Citable link http://nrs.harvard.edu/urn-3:HUL.InstRepos:12407012 Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at http:// nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of- use#LAA REVIEW ARTICLE published: 16 April 2014 doi: 10.3389/fmicb.2014.00181 BacteriophageT7 DNA polymerase – sequenase Bin Zhu* Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA Edited by: An ideal DNA polymerase for chain-terminating DNA sequencing should possess the Andrew F.Gardner, New England following features: (1) incorporate dideoxy- and other modified nucleotides at an efficiency Biolabs, USA similar to that of the cognate deoxynucleotides; (2) high processivity; (3) high fidelity in Reviewed by: the absence of proofreading/exonuclease activity; and (4) production of clear and uniform Kirk Matthew Schnorr, Novozymes A/S, Denmark signals for detection. The DNA polymerase encoded by bacteriophage T7 is naturally Samir Hamdan, King Abdullah endowed with or can be engineered to have all these characteristics. The chemically or University of Science and Technology, genetically modified enzyme (Sequenase) expedited significantly the development of DNA Saudi Arabia sequencing technology. -
Sequencing.Pdf
Sequencing. Biological sciences has a long and storied history of natural science – identifying, cataloging, and classifying organisms in different environments. Every aspect of biological sciences is being revolutionized by two dominant technologies: cheap and ubiquitous access to DNA sequencing, and cheap and ubiquitous access to high performance computing. Molecular biology was forever altered by the introduction of the polymerase chain reaction (PCR), and that invention, more than any other, has enabled the sequencing revolution. Taxonomy, one of the oldest endeavors of the biologist, is changing from char- acterizing organisms based on morphology to characterizing organisms based on DNA sequences. Revisionist taxonomic approaches are being introduced throughout the tree of life, and no branch on the tree can avoid the sequenc- ing machine. For example, microbial and viral taxonomy are being completely rewritten because of the analysis of DNA sequences (Rohwer & Edwards, 2002; Thompson et al., 2015). All aspects of ecology are being affected by the intro- duction of DNA sequencing: for example, in the marine environment the ecology of everything from the largest (Cammen et al., 2016) to the smallest (Chow & Suttle, 2015; Haggerty & Dinsdale, 2017) organisms is being revised because of sequence analysis. Evolutionary theories about plants and animals are being upended because of the introduction of genome skimming techniques and deep sequencing (Hollingsworth et al., 2016). Sanger Sequencing Two-time nobel Laureate Dr. Fred Sanger developed this sequencing technol- ogy in 1977. The approach uses terminators, dideoxynucleotide triphosphates (ddNTPs), that terminate DNA extension during replication and do not allow the strand to be synthesized further. Thus, every time a ddNTP is added to the growing DNA strand, synthesis stops and the fragement can not be made any longer. -
Experimental Quantification of Pollen with DNA Metabarcoding Using
www.nature.com/scientificreports OPEN Experimental quantifcation of pollen with DNA metabarcoding using ITS1 and trnL Sandra Baksay 1*, André Pornon1, Monique Burrus1, Jérôme Mariette2, Christophe Andalo1 & Nathalie Escaravage1 Although the use of metabarcoding to identify taxa in DNA mixtures is widely approved, its reliability in quantifying taxon abundance is still the subject of debate. In this study we investigated the relationships between the amount of pollen grains in mock solutions and the abundance of high- throughput sequence reads and how the relationship was afected by the pollen counting methodology, the number of PCR cycles, the type of markers and plant species whose pollen grains have diferent characteristics. We found a signifcant positive relationship between the number of DNA sequences and the number of pollen grains in the mock solutions. However, better relationships were obtained with light microscopy as a pollen grain counting method compared with fow cytometry, with the chloroplastic trnL marker compared with ribosomal ITS1 and with 30 when compared with 25 or 35 PCR cycles. We provide a list of recommendations to improve pollen quantifcation. Environmental DNA metabarcoding is a molecular method that consists of investigating environmental DNA samples made of complex mixtures of genomes from numerous organisms1. Due to new sequencing technologies and bioinformatics tools, metabarcoding has been increasingly used to identify taxa in environmental samples1 to monitor biodiversity2–4, to investigate ecosystem functioning5 and interaction networks6–8, in both aquatic and terrestrial ecosystems. Nevertheless, its reliability in quantitative approaches, which depend on the match between counts of high-throughput sequence reads and the amount of sampled biological material2, is still the subject of debate9,10. -
Characterization of Bacterial Communities Associated
www.nature.com/scientificreports OPEN Characterization of bacterial communities associated with blood‑fed and starved tropical bed bugs, Cimex hemipterus (F.) (Hemiptera): a high throughput metabarcoding analysis Li Lim & Abdul Hafz Ab Majid* With the development of new metagenomic techniques, the microbial community structure of common bed bugs, Cimex lectularius, is well‑studied, while information regarding the constituents of the bacterial communities associated with tropical bed bugs, Cimex hemipterus, is lacking. In this study, the bacteria communities in the blood‑fed and starved tropical bed bugs were analysed and characterized by amplifying the v3‑v4 hypervariable region of the 16S rRNA gene region, followed by MiSeq Illumina sequencing. Across all samples, Proteobacteria made up more than 99% of the microbial community. An alpha‑proteobacterium Wolbachia and gamma‑proteobacterium, including Dickeya chrysanthemi and Pseudomonas, were the dominant OTUs at the genus level. Although the dominant OTUs of bacterial communities of blood‑fed and starved bed bugs were the same, bacterial genera present in lower numbers were varied. The bacteria load in starved bed bugs was also higher than blood‑fed bed bugs. Cimex hemipterus Fabricus (Hemiptera), also known as tropical bed bugs, is an obligate blood-feeding insect throughout their entire developmental cycle, has made a recent resurgence probably due to increased worldwide travel, climate change, and resistance to insecticides1–3. Distribution of tropical bed bugs is inclined to tropical regions, and infestation usually occurs in human dwellings such as dormitories and hotels 1,2. Bed bugs are a nuisance pest to humans as people that are bitten by this insect may experience allergic reactions, iron defciency, and secondary bacterial infection from bite sores4,5. -
Sanger Sequencing – a Hands-On Simulation Background And
Sanger sequencing – a hands-on simulation Background and Guidelines for Instructor Jared Young Correspondence concerning this article should be addressed to Jared Young, Mills College 5000 MacArthur Blvd, Oakland, CA 94613 Contact: [email protected] Synopsis This hands-on simulation teaches the Sanger (dideoxy) method of DNA sequencing. In the process of carrying out the exercise, students also confront DNA synthesis, especially as it relates to chemical structure, and the stochastic nature of biological processes. The exercise is designed for an introductory undergraduate genetics course for biology majors. The exercise can be completed in around 90-minutes, which can be broken up into a 50-minute period for the simulation and a follow-up 50-minute (or less) period for discussion. This follow-up could also take place in a Teaching Assistant (TA) led section. The exercise involves interactions between student pairs and the entire class. There is an accompanying student handout with prompts that should be invoked where indicated in these instructions. Introduction Sanger sequencing is an important technique: it revolutionized the field of Genetics and is still in wide use today. Sanger sequencing is a powerful pedagogical tool well-suited for inducing multiple “aha” moments: in achieving a deep understanding of the technique, students gain a better understanding of DNA and nucleotide structure, DNA synthesis, the stochastic nature of biological processes, the utility of visible chemical modifications (in this case, fluorescent dyes), gel electrophoresis, and the connection between a physical molecule and the information it contains. Sanger sequencing is beautiful: a truly elegant method that can bring a deep sense of satisfaction when it is fully understood. -
Introduction to Bioinformatics Analysis of Metabarcoding Data
Introduction to Bioinformatics analysis of Metabarcoding data Theoretical part Alvaro Sebastián Yagüe www.sixthresearcher.com @SixthResearcher • Experimental design • Sampling • Sample processing • Sequencing • Sequence processing www.sixthresearcher.com @SixthResearcher • Experimental design • Sampling • Sample processing • Sequencing • Sequence processing www.sixthresearcher.com @SixthResearcher What do we want to sequence? Whole genome sequencing (WGS) ChIP-seq Amplicon sequencing (AS) Whole exome sequencing (WES) RNA-seq Mass spectrometry www.sixthresearcher.com @SixthResearcher How do we want to sequence? Green,E.D. (2001) Strategies for the systematic sequencing of complex www.sixthresearcher.com genomes. Nat. Rev. Genet., 2, 573–583. @SixthResearcher Metagenomics - Shotgun sequencing www.sixthresearcher.com @SixthResearcher Metagenomics - High-throughput sequencing DNA extraction DNA fragmentation and sequencing Read mapping to reference genome De novo assembly Chimeras! www.sixthresearcher.com @SixthResearcher Metabarcoding - Amplicon sequencing 1. PCR amplification and sample tagging Barcode 1 Barcode 2 Barcode 3 2. Sequencing of PCR products 3. De-multiplexing of reads Samples 1 2 3 4 5 6 Barcode 1 Barcode 2 Barcode 3 www.sixthresearcher.com @SixthResearcher Metabarcoding vs Metagenomics Kress, W. J., & Erickson, D. L. (2008). DNA barcodes: genes, genomics, and bioinformatics. Proceedings of the National Academy of Sciences of the United States of America, 105(8), 2761–2. www.sixthresearcher.com @SixthResearcher Metabarcoding vs Metagenomics http://luckylion.de/2015/10/11/dna-metabarcoding-vs-metagenomics/ www.sixthresearcher.com @SixthResearcher • Experimental design • Sampling • Sample processing • Sequencing • Sequence processing www.sixthresearcher.com @SixthResearcher ➢ Where? Variability in abundance within soil and plant. Consider vertical and horizontal distribution of fungi. ➢ When? Temporal dynamics over short and long term. For complete community census, sample across multiple seasons. -
DNA Sequencing
DNA Sequencing: Sanger Method (Dideoxynucleotide chain termination) Sanger sequencing is a DNA sequencing method in which target DNA is denatured and annealed to an oligonucleotide primer, which is then extended by DNA polymerase using a mixture of deoxynucleotide triphosphates (normal dNTPs) and chain-terminating dideoxynucleotide triphosphates (ddNTPs). ddNTPs lack the 3’ OH group to which the next dNTP of the growing DNA chain is added. Without the 3’ OH, no more nucleotides can be added, and DNA polymerase falls off. The resulting newly synthesized DNA chains will be a mixture of lengths, depending on how long the chain was when a ddNTP was randomly incorporated. Manual DNA sequencing example: • First, anneal the primer to the DNA template (must be single stranded): 5’ -GAATGTCCTTTCTCTAAG 3'-GGAGACTTACAGGAAAGAGATTCAGGATTCAGGAGGCCTACCATGAAGATCAAG-5' • Then split the sample into four aliquots including the following nucleotides: "G" tube: All four dNTPs, one of which is radiolabeled, plus ddGTP (low concentration) "A" tube: All four dNTPs, one of which is radiolabeled, plus ddATP "T" tube: All four dNTPs, one of which is radiolabeled, plus ddTTP "C" tube: All four dNTPs, one of which is radiolabeled, plus ddCTP • When a DNA polymerase (e.g. Klenow fragment) is added to the tubes, the synthetic reaction proceeds until, by chance, a dideoxynucleotide is incorporated instead of a deoxynucleotide. This is a "chain termination" event, because there is a 3' H instead of a 3' OH group. Since the synthesized DNA is labeled (classically with 35S-dATP), the products can be detected and distinguished from the template. Note that the higher the concentration of the ddNTP in the reaction, the shorter the products will be, hence, you will get sequence CLOSER to your primer.