Computational protocol: Insight into different environmental niches adaptation and allergenicity from the Cladosporium sphaerospermum genome, a common human allergy-eliciting Dothideomycetes

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Protocol publication

[…] ITS-based molecular identification was carried out using ITS region. The isolate was subjected to DNA extraction, amplification, DNA sequencing and ITS-based phylogenetic analysis was performed as previously described with slight modification. The selection of species to be included in the phylogenetic analysis was limited to species closely related to C. sphaerospermum. Complete ITS1-5.8S-ITS2 sequences for 18 C. sphaerospermum species complexes and two outgroup strains were obtained from GenBank for phylogenetic tree construction. Bayesian tree analyses were performed using MrBayes. Bayesian Markov Chain Monte Carlo (MCMC) analysis was conducted by sampling across the entire general time reversible (GTR) model space. A total of 150,000 generations were run with a sampling frequency of 100, and diagnostics were calculated for every 1,000 generations. A burn-in setting of 25% was used to discard the first 375 trees. [...] The genomic DNA extraction, sequencing, assembly, gene model prediction, and functional annotation of C. sphaerospermum UM 843 was conducted as previously described with some modifications. The 5-kb insert library was sequenced using the Illumina HiSeq 2000 system. The sequenced reads were then combined with the 500-bp Illumina sequenced reads for further processing. Genes associated with stress responses were identified by performing a local BLASTP search against a database built from the Fungal Stress Response Database (FSRD, http://internal.med.unideb.hu/fsrd) using the criteria of e-value threshold ≤1e-5, identity exceeding 50% and subject coverage exceeding 70%.The protein sequences of all current publicly-available dematiaceous Dothideomycetes genomes were downloaded from different databases to determine the orthologues in UM 843 for the orthologous genes and genome comparative analysis (). A phylogenomic tree was constructed using all proteome clusters of Dothideomycetes generated from comparative analysis with inclusion of another two proteomes from class Sordariomycetes as outgroup strains (). A total of 220 single-copy orthologous genes containing one member in each species was subjected to individual sequence alignments and removal of spurious sequences or poorly aligned regions with trimAL (with the automated option). The filtered multiple alignments were then concatenated into a superalignment with 110,781 characters. Subsequently, the best-fit substitution model was selected by running ProtTest version 3.2 with AIC calculation on the alignment. The phylogenomic tree was constructed by using MrBayes as previously described and RAxML version 7.7.9. The MCMC was run with a sampling frequency of 100 for 250,000 generations, and burn-in setting of 25%. RAxML was run with model PROTGAMMALGF to search for the best-scoring maximum likelihood tree, followed by 100 bootstrap replicates. Convergence was observed after 50 replicates using -I autoMRE option in RAxML. […]

Pipeline specifications

Software tools MrBayes, BLASTP, trimAl, ProtTest, RAxML
Databases FSRD
Application Phylogenetics
Organisms Homo sapiens
Diseases Hypersensitivity