NGS barcode sequencing in taxonomy and diagnostics, an application in \u201cCandida\u201d pathogenic yeasts with a metagenomic perspective

Claudia Colabella,Vincent Robert,Duong Vu,Gianluigi Cardinali,Joseph C Mellor,Wieland Meyer,Laura Corte,Carlo Tascini,Luca Roscini,Matteo Bassetti

doi: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.

Highlights

The regions encoding for the ribosomal DNA in yeasts are organized in an array ranging from 10 to 20 kb according to the species, including the small subunit (18S), the internal transcribed spacers (ITS1 and ITS2) and the large subunit (5S, 5.8S and 26S) (Dujon et al 2004)
In this paper we describe an innovative system of yeast VWUDLQ LGHQWL¿FDWLRQ XVLQJ QH[W JHQHUDWLRQ VHTXHQFLQJ RI amplicons covering the region spanning the ITS1 to the D1/ D2 domain of the LSU
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Summary

Introduction

The regions encoding for the ribosomal DNA in yeasts are organized in an array ranging from 10 to 20 kb according to the species, including the small subunit (18S), the internal transcribed spacers (ITS1 and ITS2) and the large subunit (5S, 5.8S and 26S) (Dujon et al 2004). In Candida albicans, the operon is 12756 bp long and the diploid genomes can contains 110 repeats in a single locus (Jones et al 2004), whereas in Candida glabrata these sequences are dispersed in two subtelomeric regions (Maleszka & ClarkɅWalker 1993, Dujon et al 2004) This difference UHÀHFWV WKH WD[RQRPLF GLVWDQFH C. albicans belongs to Debaromycetacee whereas C. glabrata belongs to Saccharomycetaceae) and the evolution, the former being a pre-genome-duplication species and the latter a postgenome-duplication species (Dujon 1996). This phenomenon can be explained by gene conversion or asymmetric crossing-over

Methods

Results

Conclusion