Single Strain High-Depth NGS Reveals High rDNA (ITS-LSU) Variability in the Four Prevalent Pathogenic Species of the Genus Candida

Abstract

Ribosomal RNA in fungi is encoded by a series of genes and spacers included in a large operon present in 100 tandem repeats, normally in a single locus. The multigene nature of this locus was somehow masked by Sanger sequencing, which produces a single sequence reporting the prevalent nucleotide of each site. The introduction of next generation sequencing led to deeper knowledge of the individual sequences (reads) and therefore of the variants between the same DNA sequences located in different tandem repeats. In this framework, NGS sequencing of the rDNA region was used to elucidate the extent of intra- and inter-genomic variation at both the strain and species level. Specifically, the use of an innovative NGS technique allowed the high-throughput high-depth sequencing of the ITS1-LSU D1/D2 amplicons of 252 strains belonging to four opportunistic yeast species of the genus Candida. Results showed the presence of a large extent of variability among strains and species. These variants were differently distributed throughout the analyzed regions with a higher concentration within the Internally Transcribed Spacer (ITS) region, suggesting that concerted evolution was not able to totally homogenize these sequences. Both the internal variability and the SNPs between strain can be used for a deep typing of the strains and to study their ecology.

When barcoding fails: Genome chimerization (admixing) and reticulation obscure phylogenetic and taxonomic relationships

“…The presence of diverse repeats can be demonstrated by cloning and sequencing ITS or D1/D2 fragments of individual rDNA repeats or by assembling complete rDNA arrays from NGS (new generation sequencing) reads. Considerable intragenomic rDNA barcode heterogeneity was detected in a few filamentous fungal species, for example in certain Amanita , Cordyceps , Laetiporus (Hughes et al, 2018; Li et al, 2013; Lindner & Banik, 2011), Hypoxylon and Pyrenopolyporus (Stadler et al, 2020) strains and, to a much lesser extent, in certain yeasts (e.g., Alper et al, 2011; Chand Dakal et al, 2016; Colabella et al, 2021; Ganley & Kobayashi, 2007; Heeger et al, 2018; Lachance et al, 2003; Roscini et al, 2018; West et al, 2014).…”

MinION Sequencing of Yeast Mock Communities To Assess the Effect of Databases and ITS-LSU Markers on the Reliability of Metabarcoding Analysis

“…It must be considered, however, that this is an unavoidable problem inherent in metabarcoding, especially when using markers of different lengths ( 29 – 31 ). Another problem linked to the current situation in fungal taxonomy, and therefore in metabarcoding, is the multigene nature of the rRNA markers and the intragenomic variability of the repeats ( 32 – 35 ). All of these factors can unbalance the relative amounts of amplicons, leading to a number of sequences not proportional to the cell densities ( 36 , 37 ), are somehow intrinsic to the biochemical procedures and to the specific nature of multigene markers of rDNA, and are hard to change as long as ITS and LSU are the most important markers in fungal taxonomy ( 6 , 38 ).…”

The enigmatic fungal genusCeraceosorusprovides a theoretical framework for studying intragenomic variation in ribosomal DNA sequences

“…This failure to directly sequence the ITS region by the Sanger method without ambiguous bases also occurs in several fungi (Pringle et al 2000;Wang and Yao 2005;Connell et al 2010;Woo et al 2010;Alper et al 2011;Harrington et al 2014;Liu et al 2015;Zhao et al 2015;Roscini et al 2018). In-depth investigations of many studies also show that intragenomic variation of the rDNA repeats exists in several fungal lineages including Agaricomycotina (Ko and Jung 2002;Wang and Yao 2005;Smith et al 2007;Lindner and Banik 2011;Vydryakova et al 2012;Lindner et al 2013;Cruz et al 2014), Glomeromycotina (Redecker et al 1999;Thiéry et al 2012;Lin et al 2014;Thiéry et al 2016;de Souza et al 2018), Mucoromycota (Woo et al 2010, Pezizomycotina (Rooney and Ward 2005;Kovács et al 2011;Li et al 2013;Naidoo et al 2013;Poczai et al 2015;Li et al 2017;Stadler et al 2020;Bradshaw et al 2023), Pucciniomycotina (Moricca et al 1996, McTaggart andAime 2018), and Saccharomycotina (James et al 2009;Dakal et al 2016;Sipiczki et al 2018;Colabella et al 2021). From these studies, we present three aspects of intragenomic variation that need to be considered: 1) variant sites (number of variant sites, from one to dozens of sites; types of variant sites, substitutions, indels or long insertions/deletions), 2) haplotypes (number of haplotypes,...…”