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      Unambiguous identification of fungi: where do we stand and how accurate and precise is fungal DNA barcoding?

      research-article
      1 , 2 , , 2 , 3 , 4 , 2 , 5 , 2 , 6 , 2 , 7 , 8 , 2 , 9 , 10 , 2 , 11 , 12 , 13 , 2 , 14 , 15 , 16 , 17 , 2 , 18 , 19 , 20 , 21 , 22 , 23 , 2 , 24 , 25 , 2 , 26 , 2 , 27 , 22 , 2 , 28 , 8 , 9 , 2 , 29 , 2 , 30 , 9 , 2 , 31 , 2 , 32 , 2 , 4
      IMA Fungus
      BioMed Central
      COX1, COX2, Oxford Nanopore technologies, PacBio, RPB2, Read placement, Species concepts, TEF1

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          Abstract

          True fungi ( Fungi) and fungus-like organisms (e.g. Mycetozoa, Oomycota) constitute the second largest group of organisms based on global richness estimates, with around 3 million predicted species. Compared to plants and animals, fungi have simple body plans with often morphologically and ecologically obscure structures. This poses challenges for accurate and precise identifications. Here we provide a conceptual framework for the identification of fungi, encouraging the approach of integrative (polyphasic) taxonomy for species delimitation, i.e. the combination of genealogy (phylogeny), phenotype (including autecology), and reproductive biology (when feasible). This allows objective evaluation of diagnostic characters, either phenotypic or molecular or both. Verification of identifications is crucial but often neglected. Because of clade-specific evolutionary histories, there is currently no single tool for the identification of fungi, although DNA barcoding using the internal transcribed spacer (ITS) remains a first diagnosis, particularly in metabarcoding studies. Secondary DNA barcodes are increasingly implemented for groups where ITS does not provide sufficient precision. Issues of pairwise sequence similarity-based identifications and OTU clustering are discussed, and multiple sequence alignment-based phylogenetic approaches with subsequent verification are recommended as more accurate alternatives. In metabarcoding approaches, the trade-off between speed and accuracy and precision of molecular identifications must be carefully considered. Intragenomic variation of the ITS and other barcoding markers should be properly documented, as phylotype diversity is not necessarily a proxy of species richness. Important strategies to improve molecular identification of fungi are: (1) broadly document intraspecific and intragenomic variation of barcoding markers; (2) substantially expand sequence repositories, focusing on undersampled clades and missing taxa; (3) improve curation of sequence labels in primary repositories and substantially increase the number of sequences based on verified material; (4) link sequence data to digital information of voucher specimens including imagery. In parallel, technological improvements to genome sequencing offer promising alternatives to DNA barcoding in the future. Despite the prevalence of DNA-based fungal taxonomy, phenotype-based approaches remain an important strategy to catalog the global diversity of fungi and establish initial species hypotheses.

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          Basic local alignment search tool.

          A new approach to rapid sequence comparison, basic local alignment search tool (BLAST), directly approximates alignments that optimize a measure of local similarity, the maximal segment pair (MSP) score. Recent mathematical results on the stochastic properties of MSP scores allow an analysis of the performance of this method as well as the statistical significance of alignments it generates. The basic algorithm is simple and robust; it can be implemented in a number of ways and applied in a variety of contexts including straightforward DNA and protein sequence database searches, motif searches, gene identification searches, and in the analysis of multiple regions of similarity in long DNA sequences. In addition to its flexibility and tractability to mathematical analysis, BLAST is an order of magnitude faster than existing sequence comparison tools of comparable sensitivity.
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            QIIME allows analysis of high-throughput community sequencing data.

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              BLAST+: architecture and applications

              Background Sequence similarity searching is a very important bioinformatics task. While Basic Local Alignment Search Tool (BLAST) outperforms exact methods through its use of heuristics, the speed of the current BLAST software is suboptimal for very long queries or database sequences. There are also some shortcomings in the user-interface of the current command-line applications. Results We describe features and improvements of rewritten BLAST software and introduce new command-line applications. Long query sequences are broken into chunks for processing, in some cases leading to dramatically shorter run times. For long database sequences, it is possible to retrieve only the relevant parts of the sequence, reducing CPU time and memory usage for searches of short queries against databases of contigs or chromosomes. The program can now retrieve masking information for database sequences from the BLAST databases. A new modular software library can now access subject sequence data from arbitrary data sources. We introduce several new features, including strategy files that allow a user to save and reuse their favorite set of options. The strategy files can be uploaded to and downloaded from the NCBI BLAST web site. Conclusion The new BLAST command-line applications, compared to the current BLAST tools, demonstrate substantial speed improvements for long queries as well as chromosome length database sequences. We have also improved the user interface of the command-line applications.
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                Author and article information

                Contributors
                r.luecking@bgbm.org
                Journal
                IMA Fungus
                IMA Fungus
                IMA Fungus
                BioMed Central (London )
                2210-6340
                2210-6359
                10 July 2020
                10 July 2020
                2020
                : 11
                : 14
                Affiliations
                [1 ]GRID grid.14095.39, ISNI 0000 0000 9116 4836, Botanischer Garten und Botanisches Museum, Freie Universität Berlin, ; Königin-Luise-Straße 6–8, 14195 Berlin, Germany
                [2 ]International Commission on the Taxonomy of Fungi, Champaign, IL USA
                [3 ]GRID grid.169077.e, ISNI 0000 0004 1937 2197, Department of Botany and Plant Pathology, , Purdue University, ; West Lafayette, IN 47907 USA
                [4 ]GRID grid.94365.3d, ISNI 0000 0001 2297 5165, National Center for Biotechnology Information, National Library of Medicine, , National Institutes of Health, ; 45 Center Drive, Bethesda, MD 20892 USA
                [5 ]GRID grid.35403.31, ISNI 0000 0004 1936 9991, Illinois Natural History Survey, , University of Illinois, ; 1816 South Oak Street, Champaign, IL 61820-6970 USA
                [6 ]GRID grid.19188.39, ISNI 0000 0004 0546 0241, Department of Plant Pathology and Microbiology, College of Bio-Resources and Agriculture, , National Taiwan University, ; Taipe City, Taiwan
                [7 ]GRID grid.507752.2, National Agriculture and Food Research Organization, Genetic Resources Center, ; 2-1-2 Kannondai, Tsukuba, Ibaraki, 305-8602 Japan
                [8 ]GRID grid.9027.c, ISNI 0000 0004 1757 3630, Department Pharmaceutical Sciences, , University of Perugia, ; Via Borgo 20 Giugno, 74, Perugia, Italy
                [9 ]GRID grid.418704.e, ISNI 0000 0004 0368 8584, Westerdijk Fungal Biodiversity Institute, ; Uppsalalaan 8, 3584 CT Utrecht, The Netherlands
                [10 ]GRID grid.4818.5, ISNI 0000 0001 0791 5666, Wageningen University and Research Centre (WUR), Laboratory of Phytopathology, ; Droevendaalsesteeg 1, 6708 PB Wageningen, The Netherlands
                [11 ]GRID grid.5329.d, ISNI 0000 0001 2348 4034, Microbiology and Applied Genomics Group, Research Area Biochemical Technology, Institute of Chemical, Environmental & Bioscience Engineering (ICEBE), TU Wien, ; Vienna, Austria
                [12 ]GRID grid.27871.3b, ISNI 0000 0000 9750 7019, Jiangsu Provincial Key Lab of Organic Solid Waste Utilization, , Nanjing Agricultural University, ; Nanjing, China
                [13 ]GRID grid.29857.31, ISNI 0000 0001 2097 4281, Department of Plant Pathology & Environmental Microbiology, , The Pennsylvania State University, ; University Park, PA 16802 USA
                [14 ]GRID grid.35937.3b, ISNI 0000 0001 2270 9879, Department of Life Sciences, The Natural History Museum, ; Cromwell Road, London, SW7 5BD UK
                [15 ]GRID grid.4903.e, ISNI 0000 0001 2097 4353, Comparative Plant and Fungal Biology, Royal Botanic Gardens, Kew, ; Surrey, TW9 3DS UK
                [16 ]GRID grid.5491.9, ISNI 0000 0004 1936 9297, Geography and Environment, , University of Southampton, ; Southampton, SO17 1BJ UK
                [17 ]GRID grid.464353.3, ISNI 0000 0000 9888 756X, Jilin Agricultural University, ; Changchun, 130118 Jilin Province China
                [18 ]GRID grid.9227.e, ISNI 0000000119573309, Key Laboratory for Plant Diversity and Biogeography of East Asia, Kunming Institute of Botany, , Chinese Academy of Science, ; Kunming, 650201 Yunnan China
                [19 ]GRID grid.411554.0, ISNI 0000 0001 0180 5757, Center of Excellence in Fungal Research, , Mae Fah Luang University, ; Chiang Rai, 57100 Thailand
                [20 ]World Agroforestry Centre, East and Central Asia, Kunming, 650201 Yunnan China
                [21 ]Mushroom Research Foundation, 128 M.3 Ban Pa Deng T. Pa Pae, A. Mae Taeng, Chiang Rai, 50150 Thailand
                [22 ]Molecular Mycology Research Laboratory, Centre for Infectious Diseases and Microbiology, Faculty of Medicine and Health, Sydney Medical School, Westmead Clinical School, Marie Bashir Institute for Infectious Diseases and Biosecurity, The University of Sydney, Westmead Hospital (Research and Education Network), Westmead Institute for Medical Research, Sydney, NSW Australia
                [23 ]GRID grid.45199.30, ISNI 0000 0001 2288 9451, Department of Health Sciences, Faculty of Science, , University of Mauritius, ; Reduit, Mauritius
                [24 ]Manaaki Whenua – Landcare Research, Private Bag 92170, Auckland, 1142 New Zealand
                [25 ]GRID grid.4903.e, ISNI 0000 0001 2097 4353, Royal Botanic Gardens, Kew, ; Surrey, TW9 3DS UK
                [26 ]GRID grid.411227.3, ISNI 0000 0001 0670 7996, Universidade Federal de Pernambuco, Centro de Biociências, Departamento de Micologia, Laboratório de Hifomicetos de Folhedo, Avenida da Engenharia, ; s/n Cidade Universitária, Recife, PE 50.740-600 Brazil
                [27 ]Royal Botanic Gardens Victoria, Birdwood Avenue, Melbourne, Victoria 3004 Australia
                [28 ]GRID grid.10939.32, ISNI 0000 0001 0943 7661, University of Tartu, ; 40 Lai Street, 51 005 Tartu, Estonia
                [29 ]GRID grid.7490.a, ISNI 0000 0001 2238 295X, Department Microbial Drugs, Helmholtz Centre for Infection Research, and German Centre for Infection Research (DZIF), partner site Hannover-Braunschweig, ; Inhoffenstrasse 7, 38124 Braunschweig, Germany
                [30 ]GRID grid.7839.5, ISNI 0000 0004 1936 9721, Institute of Ecology, Evolution and Diversity, , Goethe University, Max-von-Laue-Straße 9, 60439 Frankfurt (Main); Senckenberg Biodiversity and Climate Research Centre, ; Senckenberganlage 25, 60325 Frankfurt (Main), Germany
                [31 ]GRID grid.420081.f, ISNI 0000 0000 9247 8466, Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures, ; Braunschweig, Germany
                [32 ]GRID grid.430387.b, ISNI 0000 0004 1936 8796, Department of Plant Biology, , Rutgers University, ; New Brunswick, NJ 08901 USA
                Author information
                https://orcid.org/0000-0002-3431-4636
                https://orcid.org/0000-0001-8742-6685
                https://orcid.org/0000-0001-7300-0069
                https://orcid.org/0000-0001-8526-7721
                https://orcid.org/0000-0001-6436-3255
                https://orcid.org/0000-0002-4522-7925
                https://orcid.org/0000-0001-9085-8825
                https://orcid.org/0000-0003-2821-5268
                https://orcid.org/0000-0002-9909-0776
                https://orcid.org/0000-0002-3407-9517
                https://orcid.org/0000-0002-4453-5539
                https://orcid.org/0000-0002-8563-957X
                https://orcid.org/0000-0003-0761-9116
                https://orcid.org/0000-0002-0658-7338
                https://orcid.org/0000-0001-5920-7135
                https://orcid.org/0000-0003-2214-4972
                https://orcid.org/0000-0001-9933-8340
                https://orcid.org/0000-0001-8025-7460
                https://orcid.org/0000-0002-7284-8671
                https://orcid.org/0000-0001-7740-6875
                https://orcid.org/0000-0001-7960-2765
                https://orcid.org/0000-0002-1072-5166
                https://orcid.org/0000-0002-2680-1354
                https://orcid.org/0000-0003-1839-5322
                Article
                33
                10.1186/s43008-020-00033-z
                7353689
                32714773
                6ea82f9b-bae1-4165-b823-153d791021fd
                © The Author(s) 2020

                Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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                © The Author(s) 2020

                Plant science & Botany
                cox1,cox2,oxford nanopore technologies,pacbio,rpb2,read placement,species concepts,tef1
                Plant science & Botany
                cox1, cox2, oxford nanopore technologies, pacbio, rpb2, read placement, species concepts, tef1

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