IMA Fungus 9(1): 91-105, doi: 10.5598/imafungus.2018.09.01.07
NGS barcode sequencing in taxonomy and diagnostics, an application in “Candida” pathogenic yeasts with a metagenomic perspective
Gianluigi Cardinali‡, Laura Corte§, Luca Roscini§, Matteo Bassetti|, Carlo Tascini¶, Joseph C. Mellor#, Wieland Meyer¤, Vincent Robert, Duong Vu, Gianluigi Cardinali«‡ University of Perugia, Department of Pharmaceutical Sciences, Perugia, Italy§ University of Perugia, Microbiology Section, Department of Pharmaceutical Sciences, Italy| Santa Maria Misericordia University Hospital, Infectious Diseases Division, Udine, Italy¶ Cotugno Hospital Napoli, Infectious Diseases Division, Italy# seqWell Inc., Beverly¤ University of Sydney Centre for Infectious Diseases and Microbiology, ICPMR, Level 3, Molecular Mycology Research Laboratory, Westmead Millennium Institute, Sydney Medical School - Westmead, Westmead, Australia« University of Perugia, Department Pharmaceutical Sciences, Perugia, Italy
© Gianluigi Cardinali, Laura Corte, Luca Roscini, Matteo Bassetti, Carlo Tascini, Joseph Mellor, Wieland Meyer, Vincent Robert, Duong Vu, Gianluigi Cardinali. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY-NC 4.0), which permits to copy and distribute the article for non-commercial purposes, provided that the article is not altered or modified and the original author and source are credited. Citation:
Cardinali G, Corte L, Roscini L, Bassetti M, Tascini C, Mellor JC, Meyer W, Robert V, Vu D, Cardinali G (2018) NGS barcode sequencing in taxonomy and diagnostics, an application in “Candida” pathogenic yeasts with a metagenomic perspective. IMA Fungus 9(1): 91-105. https://doi.org/10.5598/imafungus.2018.09.01.07 |  |
Abstract
Species identification of yeasts and other Fungi is currently carried out with Sanger sequences of selected molecular markers, mainly from the ribosomal DNA operon, characterized by hundreds of tandem repeats of the 18S, ITS1, 5.8S, ITS2 and LSU loci. The ITS region has been recently proposed as a primary barcode marker making this region the most used one in taxonomy, phylogeny and diagnostics. The introduction of NGS is providing tools of high efficacy and relatively low cost to amplify two or more markers simultaneously with great sequencing depth. However, the presence of intra-genomic variability between the repeats requires specific analytical procedures and pipelines. In this study, 286 strains belonging to 11 pathogenic yeasts species were analysed with NGS of the region spanning from ITS1 to the D1/D2 domain of the LSU encoding ribosomal DNA. Results showed that relatively high heterogeneity can hamper the use of these sequences for the identification of single strains and even more of complex microbial mixtures. These observations point out that the metagenomics studies could be affected by species inflection at levels higher than currently expected.
Keywords
identification, ITS, LSU, Next generation sequencing, Sanger