Computational protocol: Multilocus variable-number tandem-repeat analysis of clinical isolates of Aspergillus flavus from Iran reveals the first cases of Aspergillus minisclerotigenes associated with human infection

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Protocol publication

[…] Phylogenetic analysis of Iranian isolates grouping with A. minisclerotigenes was done using partial β-tubulin and calmodulin gene sequences. Sequences were amplified from selected isolates using MyTaq Red Master Mix (Bioline, UK) according to manufacturer’s instructions and previously published primers for β-tubulin (bt2a and bt2b, []) and calmodulin (cf1 and cf4 []). Sequences were edited in Geneious Pro 6.1.6 (created by Biomatters; available at http://www.geneious.com/) and were aligned with published Aspergillus section flavi sequences []. Phylogenetic analysis was undertaken using maximum likelihood with the PAUP 4.0 plug-in for Geneious. 1000 replicates were used to determine bootstrap support values. Accession numbers are provided in on the relevant phylogenetic trees. All taxa information, matrices and trees have been made available through TreeBASE at the following URL: http://purl.org/phylo/treebase/phylows/study/TB2:S15946.Mating type analysis was performed on all Group II/A. minisclerotigenes-like isolates in our collection along with selected type strains. Mating type was determined by amplification using MAT1-1 and MAT1-2 specific primers (M1F and M1R, M2F and M2R; []) in separate reactions. PCR amplification was performed using MyTaq Red Master Mix (Bioline, UK) according to manufacturer’s instructions. [...] Initial analysis of the MLV data used MICROSAT version 1.4 to calculate pairwise population distances using the proportional shared allele distance measure (DPs) []. Null alleles were scored as missing data. Pairwise distances were used to construct dendrograms using the Neighbour-Joining algorithm [] available in the program PHYLIP 3.5c Bootstrap analyses were performed in MICROSAT with 1,000 replications. A. parasiticus reference strains were used as the outgroup.To further analyze the structure of the Iranian population and to integrate this with other isolates a network-based approach was used using goeBURST with Minimum Spanning Tree implemented in PHYLOVIZ 1.0 [] (available at http://goeburst.phyloviz.net/#Software). Isolates were assigned multilocus variable-number tandem-repeat (MLV) genotypes based on their VNTR data (Table  ) and these were integrated with taxonomic, geographic, clinical and toxin data to visualize and assess the relationships among strains. […]

Pipeline specifications

Software tools Geneious, PAUP*, PhyloWS, PHYLIP, BURST, PHYLOViZ
Databases TreeBASE
Applications Phylogenetics, WGS analysis
Organisms Aspergillus flavus, Homo sapiens
Diseases Infection
Chemicals Aflatoxins