Computational protocol: The Black Yeast Exophiala dermatitidis and Other Selected Opportunistic Human Fungal Pathogens Spread from Dishwashers to Kitchens

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Protocol publication

[…] Identification of the yeast was based on amplification and sequencing of the large subunit ribosomal DNA sequences (LSU; partial 28S rDNA, D1/D2 domains), using the NL1 and NL4 primer set []. A fragment of the rDNA that included internal transcribed spacer (ITS) region 1, 5.8S rDNA, and ITS2 was amplified and sequenced for identification of Exophiala spp. and other filamentous fungi, using the ITS5 and ITS4 primer set []. For identification of Aspergillus and Penicillium strains, the partial β-tubulin gene (benA) was amplified and sequenced with the T1 and T22 primers []. Fusarium strains were identified using nuclear translation elongation factor 1-alpha (tef) sequences, amplified with the EF1 and EF2 primers [].The ITS, LSU, benA and tef nucleotide sequences were determined by direct PCR sequencing, performed by Microsynth AG, Switzerland. BigDye terminator cycle sequencing kits were used in the sequence reactions (Applied Biosystems, Foster City, CA, USA). The sequences were obtained using an ABI Prism 3700 Big Dye Sequencer (Applied Biosystems). The sequences were assembled using FinchTV 1.4 (Geospiza, PerkinElmer, Inc.), and automatically and manually aligned using the Molecular Evolutionary Genetics Analysis (MEGA) software, version 6.06 []. The assembled DNA sequences were examined using the BLAST software of the National Centre for Biotechnology Information (NCBI) database, and were compared to the appropriate sequences of the reference and type strains.The genotypes of E. dermatitidis were defined from sequences obtained according to Matos et al. (2003) []. Determination of E. phaeomuriformis genotypes was made based on Zalar et al. (2011) [].Validation of the presence of E. dermatitidis from genomic DNA in an air sample was conducted by PCR amplification with the specific primers Eder1F (5´-TCT GGT CGA GCG TTT CCG-3´) and Eder1R (5´-AAA CCG ATA CGT GCT CAG TGA-3´), using an established protocol designed by Novak Babič et al. (2013) []. [...] An artificial community DNA template was obtained by combining aliquots of the 30 individual genomic DNA extracts from fresh biofilms. The amount of DNA of this representative biofilm DNA sample that was used for downstream sequencing was 50 ng. A single sample of total DNA was extracted from a dried biofilm, with an amount of 3.6 ng. Equal amount of template DNA was used for PCR reaction in both samples. Both samples were sequenced separately, where ITS2 of the nuclear ribosomal coding cistron was targeted with the ITS3 and ITS4 primers []. The amplicon was sequenced by Microsynth AG (Balgach, Switzerland) using the Roche 454 pyrosequencing platform. The sequences were processed bioinformatically using QIIME []. Reads with less than 60% similarity to any sequence in the reference dataset were discarded. Sequences with quality below 25 or shorter than 250 bp were removed. Chimeric sequences were identified using the UCHIME algorithm [], and were discarded. The linker and reverse primer sequences were trimmed. The maximum allowed number of homopolymers in a single fungal sequence was set to eight. All of the sequences were clustered into Operational Taxonomic Units (OTUs) based on their sequence similarities. This was carried out by subsampled open reference clustering against the dynamic reference set of the UNITE database (version 6). When the sequences were clustered at 97% sequence similarity, those belonging to the same cluster were interpreted as the same species. The OTUs were identified by similarity to reference sequences (minimum similarity, 97%), and de-novo by clustering the remaining sequences with each other (minimum similarity, 97%). The set of representative sequences was assigned to taxonomic identities using the UNITE/QIIME ITS reference OTUs. The reference sequence set represented a clustered version (sequence similarity, 97%) of all of the fungal rDNA ITS sequences in the current UNITE+INSD databases (International Nucleotide Sequence Databases: NCBI, EMBL, DDBJ). […]

Pipeline specifications

Software tools QIIME, UCHIME
Application 16S rRNA-seq analysis
Organisms Saccharomyces cerevisiae, Exophiala dermatitidis, Homo sapiens, Fungi, Exophiala oligosperma, Candida parapsilosis
Diseases Mycoses