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      Dynamic time warping assessment of high-resolution melt curves provides a robust metric for fungal identification

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          Abstract

          Fungal infections are a global problem imposing considerable disease burden. One of the unmet needs in addressing these infections is rapid, sensitive diagnostics. A promising molecular diagnostic approach is high-resolution melt analysis (HRM). However, there has been little effort in leveraging HRM data for automated, objective identification of fungal species. The purpose of these studies was to assess the utility of distance methods developed for comparison of time series data to classify HRM curves as a means of fungal species identification. Dynamic time warping (DTW), first introduced in the context of speech recognition to identify temporal distortion of similar sounds, is an elastic distance measure that has been successfully applied to a wide range of time series data. Comparison of HRM curves of the rDNA internal transcribed spacer (ITS) region from 51 strains of 18 fungal species using DTW distances allowed accurate classification and clustering of all 51 strains. The utility of DTW distances for species identification was demonstrated by matching HRM curves from 243 previously identified clinical isolates against a database of curves from standard reference strains. The results revealed a number of prior misclassifications, discriminated species that are not resolved by routine phenotypic tests, and accurately identified all 243 test strains. In addition to DTW, several other distance functions, Edit Distance on Real sequence (EDR) and Shape-based Distance (SBD), showed promise. It is concluded that DTW-based distances provide a useful metric for the automated identification of fungi based on HRM curves of the ITS region and that this provides the foundation for a robust and automatable method applicable to the clinical setting.

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          ITS as an environmental DNA barcode for fungi: an in silico approach reveals potential PCR biases

          Background During the last 15 years the internal transcribed spacer (ITS) of nuclear DNA has been used as a target for analyzing fungal diversity in environmental samples, and has recently been selected as the standard marker for fungal DNA barcoding. In this study we explored the potential amplification biases that various commonly utilized ITS primers might introduce during amplification of different parts of the ITS region in samples containing mixed templates ('environmental barcoding'). We performed in silico PCR analyses with commonly used primer combinations using various ITS datasets obtained from public databases as templates. Results Some of the ITS primers, such as ITS1-F, were hampered with a high proportion of mismatches relative to the target sequences, and most of them appeared to introduce taxonomic biases during PCR. Some primers, e.g. ITS1-F, ITS1 and ITS5, were biased towards amplification of basidiomycetes, whereas others, e.g. ITS2, ITS3 and ITS4, were biased towards ascomycetes. The assumed basidiomycete-specific primer ITS4-B only amplified a minor proportion of basidiomycete ITS sequences, even under relaxed PCR conditions. Due to systematic length differences in the ITS2 region as well as the entire ITS, we found that ascomycetes will more easily amplify than basidiomycetes using these regions as targets. This bias can be avoided by using primers amplifying ITS1 only, but this would imply preferential amplification of 'non-dikarya' fungi. Conclusions We conclude that ITS primers have to be selected carefully, especially when used for high-throughput sequencing of environmental samples. We suggest that different primer combinations or different parts of the ITS region should be analyzed in parallel, or that alternative ITS primers should be searched for.
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            High-resolution DNA melting analysis for simple and efficient molecular diagnostics.

            High-resolution melting of DNA is a simple solution for genotyping, mutation scanning and sequence matching. The melting profile of a PCR product depends on its GC content, length, sequence and heterozygosity and is best monitored with saturating dyes that fluoresce in the presence of double-stranded DNA. Genotyping of most variants is possible by the melting temperature of the PCR products, while all variants can be genotyped with unlabeled probes. Mutation scanning and sequence matching depend on sequence differences that result in heteroduplexes that change the shape of the melting curve. High-resolution DNA melting has several advantages over other genotyping and scanning methods, including an inexpensive closed tube format that is homogenous, accurate and rapid. Owing to its simplicity and speed, the method is a good fit for personalized medicine as a rapid, inexpensive method to predict therapeutic response.
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              Candida orthopsilosis and Candida metapsilosis spp. nov. to replace Candida parapsilosis groups II and III.

              Two new species, Candida orthopsilosis and C. metapsilosis, are proposed to replace the existing designations of C. parapsilosis groups II and III, respectively. The species C. parapsilosis is retained for group I isolates. Attempts to construct a multilocus sequence typing scheme to differentiate individual strains of C. parapsilosis instead revealed fixed DNA sequence differences between pairs of subgroups in four genes: COX3, L1A1, SADH, and SYA1. PCR amplicons for sequencing were obtained for these four plus a further seven genes from 21 group I isolates. For nine group II isolates, PCR products were obtained from only 5 of the 11 genes, and for two group III isolates PCR products were obtained from a different set of 5 genes. Three of the PCR products from group II and III isolates differed in size from the group I products. Cluster analysis of sequence polymorphisms from COX3, SADH, and SYA1, which were common to the three groups, consistently separated the isolates into three distinct sets. All of these differences, together with DNA sequence similarities <90% in the ITS1 sequence, suggest the subgroups should be afforded species status. The near absence of DNA sequence variability among isolates of C. parapsilosis and relatively high levels of sequence variability among isolates of C. orthopsilosis suggest that the former species may have evolved very recently from the latter.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                6 March 2017
                2017
                : 12
                : 3
                : e0173320
                Affiliations
                [1 ]Dermatology Department, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou, China
                [2 ]Institute for Microbiology and Parasitology, University Sts Cyril and Methodius, Skopje, Macedonia
                [3 ]Department of Microbiology & Immunology, Georgetown University, Washington, DC, United States of America
                [4 ]Walter Reed Army Institute of Research (WRAIR), Silver Spring, MD, United States of America
                Institute of Microbiology, SWITZERLAND
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                • Conceptualization: WAF.

                • Formal analysis: WAF.

                • Funding acquisition: RAC WAF.

                • Investigation: SL GM SP JL WAF.

                • Methodology: WAF.

                • Project administration: WAF.

                • Resources: DL JL RAC WAF.

                • Supervision: RAC WAF.

                • Validation: WAF.

                • Visualization: WAF.

                • Writing – original draft: SL WAF.

                • Writing – review & editing: SL GM JL RAC WAF.

                Author information
                http://orcid.org/0000-0002-4573-9955
                Article
                PONE-D-16-46383
                10.1371/journal.pone.0173320
                5338801
                28264030
                80ed6b7f-515a-4710-aa3a-82e49bd942ee
                © 2017 Lu et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 22 November 2016
                : 17 February 2017
                Page count
                Figures: 9, Tables: 2, Pages: 21
                Funding
                The authors received no specific funding for this work.
                Categories
                Research Article
                Biology and Life Sciences
                Organisms
                Fungi
                Yeast
                Candida
                Candida Albicans
                Biology and Life Sciences
                Microbiology
                Medical Microbiology
                Microbial Pathogens
                Fungal Pathogens
                Candida Albicans
                Medicine and Health Sciences
                Pathology and Laboratory Medicine
                Pathogens
                Microbial Pathogens
                Fungal Pathogens
                Candida Albicans
                Biology and Life Sciences
                Mycology
                Fungal Pathogens
                Candida Albicans
                Research and Analysis Methods
                Experimental Organism Systems
                Yeast and Fungal Models
                Candida Albicans
                Research and Analysis Methods
                Database and Informatics Methods
                Biological Databases
                Sequence Databases
                Research and Analysis Methods
                Database and Informatics Methods
                Bioinformatics
                Sequence Analysis
                Sequence Databases
                Research and analysis methods
                Database and informatics methods
                Bioinformatics
                Sequence analysis
                DNA sequence analysis
                Engineering and Technology
                Measurement
                Distance Measurement
                Biology and Life Sciences
                Organisms
                Fungi
                Yeast
                Biology and Life Sciences
                Molecular Biology
                Molecular Biology Techniques
                Sequencing Techniques
                Nucleotide Sequencing
                Research and Analysis Methods
                Molecular Biology Techniques
                Sequencing Techniques
                Nucleotide Sequencing
                Biology and Life Sciences
                Mycology
                Fungal Classification
                Biology and Life Sciences
                Molecular Biology
                Molecular Biology Techniques
                Artificial Gene Amplification and Extension
                Polymerase Chain Reaction
                Research and Analysis Methods
                Molecular Biology Techniques
                Artificial Gene Amplification and Extension
                Polymerase Chain Reaction
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